Clostridium Difficile Antibodies

ABSTRACT

Compositions and methods for the treatment or prevention of  Clostridium difficile  infection in a subject are provided. The compositions comprise antibodies to  Clostridium difficile  toxin A. The methods provide for administering the antibodies to a subject in an amount effective to reduce or eliminate or prevent relapse from  Clostridium difficile  bacterial infection.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.61/526,031 filed Aug. 22, 2011, the disclosure of which is incorporatedherein by reference in its entirety.

FIELD

The invention relates to monoclonal antibodies to Clostridium difficiletoxin A. The invention further relates to compositions and methods forthe treatment or prevention of infection by the bacteria, Clostridiumdifficile, in a vertebrate subject. Methods are provided foradministering antibodies to the vertebrate subject in an amounteffective to reduce, eliminate, or prevent relapse from infection.Methods for the treatment or prevention of Clostridium difficileinfection in an organism are provided.

BACKGROUND

Clostridium difficile (C. difficile) is a common nosocomial pathogen anda major cause of morbidity and mortality among hospitalized patientsthroughout the world. Kelly et al., New Eng. J. Med., 330:257-62, 1994.The increased use of broad spectrum antibiotics and the emergence ofunusually virulent strains of C. difficile have lead to the idea thatvaccines may be well suited to reduce disease and death associated withthis bacterium. C. difficile has few traditional antibiotic options andfrequently causes a recurring disease (25% of cases). C. difficileclaims about 20,000 lives in the USA alone per year and causes around500,000 confirmed infections. Recently, more virulent strains of C.difficile have emerged that produce more toxin such as the B1/NAB1/027strain, which also has a decreased susceptibility to metronidazole.Outbreaks of C. difficile have necessitated ward and partial hospitalclosure. With the increasing elderly population and the changingdemographics of the population, C. difficile is set to become a majorproblem in the 21^(st) century. The spectrum of C. difficile diseaseranges from asymptomatic carriage to mild diarrhea to fulminantpseudomembranous colitis.

C. difficile has a dimorphic lifecycle whereby it exists both as aninfectious and tough spore form and a metabolically activetoxin-producing vegetative cell. C. difficile-associated disease (CDAD)is believed to be caused by the vegetative cells and more specificallythe actions of two toxins, enterotoxin toxin A and cytotoxin toxin B.Vaccines and therapy for C. difficile have been to date focused upon thetoxins (A and B), toxoids of A and B, recombinant fragments of A and B,and vegetative cell surface layer proteins (SLPAs).

Toxin A is a high-molecular weight protein that possesses multiplefunctional domains. The toxin is broken up into 4 functional domains: anamino-terminal glucosyltransferase that modifies Rho-like GTPasesleading to cytoskeletal dysregulation in epithelial cells, anautocatalytic cysteine protease domain, a hydrophobic membrane-spanningsequence, and a highly repetitive carboxy-terminal host-cell bindingdomain. The carboxy terminal domain anchors the toxin to the host cellcarbohydrate receptors on intestinal epithelial cells which initiatesthe internalization process thereby delivering the amino-terminalenzymatic domains to the cytoplasm of the target cells. The delivery ofthe enzymatic domain and glucosyltransferase activity leads to diarrheaand inflammation due to the apoptotic cell death of the intoxicatedcells.

Many studies have shown the importance of antibodies against the toxinsin affecting the disease outcome. Studies have also shown thecorrelation between serum anti-toxinA antibodies with protection fromCDAD and relapse. These studies have led to the creation of toxin mAbtherapies for CDAD.

Despite these advances, there is an unmet need for effective treatmentand/or prevention of C. difficile associated infections includingprevention from relapse of CDAD. The present invention provides mouseand humanized antibodies to toxin A to satisfy these and other needs.

SUMMARY

The present invention provides for antibodies, or antigen-bindingportions thereof, that bind to Clostridium difficile (C. difficile)toxin A. The antibody or antigen-binding portion thereof may bind tofragment 4 of C. difficile toxin A.

In one embodiment, the present invention provides for an isolatedmonoclonal antibody, or an antigen-binding portion thereof, comprising aheavy chain region and a light chain region, wherein the heavy chainregion comprises three complementarity determining regions (CDRs), CDR1,CDR2 and CDR3, having amino acid sequences about 80% to about 100%homologous to the amino acid sequences set forth in SEQ ID NOs: 29, 30and 31, respectively, and wherein the light chain region comprises threeCDRs, CDR1, CDR2 and CDR3, having amino acid sequences about 80% toabout 100% homologous to the amino acid sequences set forth in SEQ IDNOs: 21, 22 and 23, respectively.

Also provided is an isolated monoclonal antibody, or an antigen-bindingportion thereof, that binds to C. difficile toxin A and comprises aheavy chain region, wherein the heavy chain region comprises three CDRs,CDR1, CDR2 and CDR3, having amino acid sequences about 80% to about 100%homologous to the amino acid sequences set forth in SEQ ID NOs: 29, 30and 31, respectively.

The present invention further provides for an isolated monoclonalantibody, or an antigen-binding portion thereof, that binds to C.difficile toxin A and comprises a light chain region, wherein the lightchain region comprises three CDRs, CDR1, CDR2 and CDR3, having aminoacid sequences about 80% to about 100% homologous to the amino acidsequences set forth in SEQ ID NOs: 21, 22 and 23, respectively.

The antibody or antigen-binding portion thereof may have a dissociationconstant (K_(D)) of less than about 1×10⁻¹¹ M. The antibody orantigen-binding portion thereof may be humanized or chimeric.

In one embodiment, the heavy chain region of the antibody orantigen-binding portion thereof comprises an amino acid sequence about80% to about 100% homologous to the amino acid sequence set forth in SEQID NO: 89; the light chain region of the antibody or antigen-bindingportion thereof comprises an amino acid sequence about 80% to about 100%homologous to the amino acid sequence set forth in SEQ ID NO: 91.

In another embodiment, the heavy chain region of the antibody orantigen-binding portion thereof comprises an amino acid sequence about80% to about 100% homologous to the amino acid sequence set forth in SEQID NO: 93; the light chain region of the antibody or antigen-bindingportion thereof comprises an amino acid sequence about 80% to about 100%homologous to the amino acid sequence set forth in SEQ ID NO: 95.

The antibody or antigen-binding portion thereof may be the following:(a) a whole immunoglobulin molecule; (b) an scFv; (c) a Fab fragment;(d) an F(ab′)2; and (e) a disulfide linked Fv.

The antibody or antigen-binding portion thereof may comprise at leastone constant domain selected from the group consisting of: a) an IgGconstant domain; and (b) an IgA constant domain.

One embodiment of the present invention provides for an isolatedmonoclonal antibody or an antigen-binding portion thereof, that binds toC. difficile toxin A and comprises a heavy chain variable region,wherein the heavy chain variable region comprises an amino acid sequenceabout 80% to about 100% homologous to the amino acid sequence set forthin SEQ ID NOs: 12, 28, 44 or 60.

Another embodiment of the present invention provides for an isolatedmonoclonal antibody, or an antigen-binding portion thereof, that bindsto C. difficile toxin A and comprises a light chain variable region,wherein the light chain variable region comprises an amino acid sequenceabout 80% to about 100% homologous to the amino acid sequence set forthin SEQ ID NOs: 4, 20, 36 or 52.

Yet another embodiment of the present invention provides for an isolatedmonoclonal antibody, or an antigen-binding portion thereof, wherein theantibody, or antigen-binding portion thereof, binds to the same epitopeof C. difficile toxin A recognized by an antibody comprising a heavychain variable region and a light chain variable region having aminoacid sequences about 80% to about 100% homologous to the amino acidsequences set forth in SEQ ID NOs: 28 and 20, respectively.

Also encompassed by the present invention are an antibody produced byhybridoma designated CAN20G2 and the hybridoma designated CAN20G2.

The present invention provides for an isolated monoclonal antibody, oran antigen-binding portion thereof, wherein, in an in vivo toxin Achallenge experiment, when the antibody, or an antigen-binding portionthereof, is administered to a mammal at a dosage ranging from about 8mg/kg body weight to about 13 mg/kg body weight about 24 hours beforethe mammal is exposed to greater than about 100 ng of C. difficile toxinA, the chance of survival for the mammal is greater than about 80%within about 7 days.

Also encompassed by the present invention is an isolated monoclonalantibody, or an antigen-binding portion thereof, wherein the antibody,or antigen-binding portion thereof, at a concentration ranging fromabout 4 μM to about 17 μM, neutralizes greater than about 40% of about150 ng/ml C. difficile toxin A in an in vitro neutralization assay.

The present invention provides for an isolated nucleic acid encoding apeptide comprising an amino acid sequence about 80% to about 100%homologous to the amino acid sequence set forth in SEQ ID NOs: 12, 28,44, 60, 4, 20, 36 or 52. The present invention also provides for anisolated nucleic acid comprising a nucleic acid sequence about 80% toabout 100% homologous to the nucleic acid sequence set forth in SEQ IDNOs: 68, 69, 70, 71, 72, 73, 74 or 75. Also provided is a cellcomprising any of these nucleic acids. The cell can be a bacterial cellor a eukaryotic cell, such as a mammalian cell. Non-limiting examples ofthe cells include COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2,SP2/0, HeLa, myeloma or lymphoma cells.

The present invention provides for a composition comprising the antibodyor antigen-binding portion thereof and at least one pharmaceuticallyacceptable carrier.

The present invention provides for a method of preventing or treating C.difficile-associated disease comprising administering to a subject aneffective amount of the present antibody or antigen-binding portionthereof. The antibody or antigen-binding portion thereof may beadministered intravenously, subcutaneously, intramuscularly ortransdermally. The method may contain another step of administering tothe subject a second agent. For example, the second agent may be adifferent antibody or fragment thereof, or may be an antibiotic such asvancomycin, metronidazole or fidaxomicin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a standardized ELISA showing the reactivity of purifiedmurine mAbs on Clostridium difficile toxin A.

FIG. 2 is an ELISA showing the binding activity of purified 1 μg/mlCAN19 mAbs on toxin A (ToxA) and toxin A fragment 4 (ToxAF4). ToxB istoxin B; ToxBF4 is toxin B fragment 4.

FIG. 3 is an ELISA assay showing the binding activity of purified 1μg/ml murine CAN20 mAbs on toxin A and toxin A fragment 4.

FIG. 4 shows a Western immunoblot of Purified Murine CAN19 mAbs (0.5μg/ml). Lane 1: Toxin A; Lane 2: Toxoid A; Lane 4: Toxin A Fragment 4;Lane 5: Toxin B; Lane 7: Toxin B Fragment 4; Lane 8: PilF (negativecontrol). Expected sizes: Toxin A (308 kDa); Toxin A Fragment 4 (114kDa); Toxin B (280 kDa).

FIG. 5 shows a Western blot of Purified CAN20 clones (1 μg/ml). Blot Awas probed with CAN20G1, blot B was probed with CAN20G2, blot C wasprobed with CAN20G5, and blot D was probed with Can20G8. (Lane 1: ToxinA (308 kDa); Lane 2: Toxin A Fragment 4 (114 kDa); Lane3: Toxin B (280kDa); Lane4: tetanus toxoid).

FIG. 6a is an epitope binning graph showing biotinylated CAN20G1antibody binding to SA (streptavidin) biosensor. The bound antibody isthen incubated with free Toxin A and free CAN20G1. The CAN20G1-Toxin Acomplex is again incubated with free antibody. A large nm shift inwavelength will indicate binding of the analyte indicating that CAN20G1and the free antibody have different epitopes. 1, Biotinylated CAN20G1to SA biosensors. 2, Free whole toxin A forming complex with CAN20G1. 3,Free CAN20G1 associating with biotinylated CAN20G1-Toxin A complex. 4,Association sample curves. 5, Dissociation step.

FIG. 6b is a graph showing the final three steps (3-5) of the fullprogram. A large nm shift in wavelength will indicate binding of theanalyte indicating that CAN20G1 and the free antibody have differentepitopes. In this case, only CDA1 (Merck anti-toxin A mAb used as acontrol) had a significant nm shift in wavelength demonstrating thatCDA1 binds to a different epitope while CAN20G1, G2, G5, and G8 bind tothe same epitope bin as CAN20G1.

FIG. 7 is a bar graph showing the effects of C. difficile toxin A onmouse survival and the efficacy of the CAN19 mAbs against the toxin Achallenge.

FIG. 8 is a bar graph showing the effects of C. difficile toxin A onmouse survival and the efficacy of the CAN19 and CAN20 mAbs againsttoxin A challenge.

FIG. 9 is a bar graph showing the effects of C. difficile toxin A onmouse survival and the efficacy of the murine CAN20G2 mAb at full doseand half dose against toxin A challenge.

FIG. 10 shows primers used for V gene amplification from RNA. Thedegenerate base symbols are IUPAC (International union of pure andapplied chemistry) codes for representing degenerate nucleotide sequencepatterns.

FIG. 11 shows V-gene sequencing results for muCAN20G2 that includes bothVH and VL sequences from the muCAN20G2 parental clones.

FIG. 12 shows alignment of muCAN20G2 v-regions with the closest humangermline v-region. The human germlines were used as acceptor frameworksfor humanization.

FIGS. 13a and 13b show CDR-huCAN20G2 design. The closest matching humanframeworks are IGHV7-4-1*02 and IGKV1-39*01. The CDRs (IMGT Numbering)of the muCAN20G2 were inserted into the human framework. FIG. 13A showsthe heavy chain variable region, including both nucleic acid sequenceand amino acid sequence. FR1, FR2 and FR3 are from IGHV7-4-1*02; FR4 isfrom IGHJ6*01.

FIG. 13B shows the light (kappa) chain variable region, including bothnucleic acid sequence and amino acid sequence. FR1, FR2 and FR3 are fromIGKV1-39*01; FR4 is from IGKJ4*01.

FIGS. 14a and 14b show HE-huCAN20G2 Design. Resurfaced and alteredcodons are in bold. The nucleotide sequence was translated to ensurecorrect frame.

FIG. 14A shows the heavy chain variable region, including both nucleicacid sequence and amino acid sequence. FIG. 14B shows the light (kappa)chain variable region, including both nucleic acid sequence and aminoacid sequence.

FIG. 15 shows the HE-huCAN20G2 Heavy Chain. Resurfaced and alteredcodons are in bold. After v-region design, an IgG1 constant region wasadded. The introns were removed and the nucleotide sequence wastranslated to ensure correct frame.

FIG. 16 shows HE-huCAN20G2 Kappa Chain. Resurfaced and altered codonsare in bold. After v-region design, a Kappa constant region was added.The introns were removed and the nucleotide sequence was translated toensure correct frame.

FIG. 17 shows AVA-huCAN20G2 kappa V-region alignment. The Avastin kappav-region was aligned to the IMGT domain directory and identified theclosest germline v-region. IGKV1D-33-01 was used as the acceptorframework for the AVA mAb design.

FIG. 18 shows AVA-huCAN20G2. The Avastin kappa v-region was aligned tothe IMGT domain directory and identified the closest germline v-region.After analysis and design, a kappa constant region was added. Aspreviously, the constant regions contain introns. For the AVA-huCAN20G2heavy chain, the previously designed and resurfaced HE-huCAN20G2 heavychain was used. FR1, FR2 and FR3 are from IGKV1D-33-01; FR4 is fromIGKJ1-01.

FIGS. 19a and 19b show chimeric CAN20G2. Murine V-regions were designedwith human constant regions. The introns were removed and the nucleotidesequence was translated to ensure correct frame. FIG. 19A shows theheavy chain, including both nucleic acid sequence and amino acidsequence. FIG. 14B shows the light (kappa) chain, including both nucleicacid sequence and amino acid sequence.

FIG. 20a shows neutralization data for purified human CAN20G2 clones at150 ng/ml depicted as a bar graph.

FIG. 20b shows neutralization data for purified human CAN20G2 clones at250 ng/ml depicted as a bar graph.

FIG. 21a shows ELISA to screen transfection supernatant for expressedhuman Can20G2 mAbs binding to toxin A at 45 minutes.

FIG. 21b shows ELISA to screen transfection supernatant for expressedhuman Can20G2 mAbs binding to toxin A fragment 4 at 45 minutes.

FIG. 21c shows ELISA to screen transfection supernatant for expressedhuman Can20G2 mAbs binding to toxin A at 60 minutes.

FIG. 21d shows an ELISA to screen transfection supernatant for expressedhuman Can20G2 mAbs binding to toxin A fragment 4 at 60 minutes.

FIG. 22 shows SDS-PAGE of purified human CAN20G2 clones.

FIG. 23 shows Western blot analysis of purified human CAN20G2 clones. AnSDS-page gel was run with tetanus toxoid, whole toxin A, toxin Afragment 4 and BSA. The gel was transferred to nitrocellulose membraneand probed with each of the human CAN20G2 mAbs (1 μg/ml). (Lane 1: ToxinA; Lane 2: Toxin A Fragment 4; Lane 3: tetanus toxoid; Lane 4: BSA).

FIGS. 24a and 24b show healthy donor T cell proliferation responses totest antibodies, CDR-huCAN20G2 (FIG. 24A) and HE-huCAN20G2 (FIG. 24B),on days 5, 6, 7, and 8 after incubation. Proliferation responses with anSI≧2.00 (indicated by dotted line) that were significant (p<0.05) usingan unpaired, two sample student's t test were considered positive. Foreach donor, the bars from left to right represent day 5, day 6, day 7and day 8, respectively.

FIG. 25 shows the number of positive T cell proliferation responses toantibodies CDR-huCAN20G2 (C001) and HE-huCAN20G2 (H001) detected at fourtime points.

FIG. 26 shows healthy donor T cell IL-2 ELISpot responses to testantibodies, CDR-huCAN20G2 (C001) and HE-huCAN20G2 (H001). PBMCs wereused to assess IL-2 secretion in response to stimulation with the twoantibodies during an 8-day incubation. T cell responses with an SI≧2.00that were significant (p<0.05) using an unpaired, two sample student's ttest were scored positive. Borderline responses (significant p<0.05 withSI≧1.90) was shown (*).

FIG. 27 shows the comparison of HE-huCAN20G2 (“HE-CAN20G2”),CDR-huCAN20G2 (“CDR-CAN20G2”) and CDA1 (Merck/Medarex) anti-C. difficiletoxin A (anti-TcdA) mAbs tested at a low dose of 0.05 mg/mouse. Efficacyof mAbs is presented as the percentages of survival compared to controlanimals (TcdA/PBS). *Fisher exact test for statistical significance.

FIG. 28 shows the effect of humanized CAN20G2 mAbs, HE-huCAN20G2,CDR-huCAN20G2 in comparison with CDA1 on survival over time followingTcdA challenge. The effect of mAbs at low dose of Ab (0.05 mg) or PBSalone (control) on survival related to time after TcdA challenge isdepicted. The percent survival of animals in each group post TcdAchallenge at the indicated time points (hrs) is shown in the graph.

FIGS. 29a and 29b show PK study data of humanized antibodiesCDR-huCAN20G2 (FIG. 29a ) and HE-huCAN20G2 (FIG. 29b ) in rats.

DETAILED DESCRIPTION

The present invention provides for compositions and methods for theprevention or treatment of Clostridium difficile bacterial infection orbacterial carriage. The compositions contain antibodies (or anantigen-binding portion thereof) that recognize toxin A of C. difficile,including mouse monoclonal antibodies, humanized antibodies, chimericantibodies, or antigen-binding portions of any of the foregoing. Theseantibodies (or antigen-binding portion thereof) can neutralize toxin Ain vitro and in vivo, and/or inhibit binding of toxin A to mammaliancells. Therefore, the present antibodies or antigen-binding portionthereof can be used in passive immunization to prevent or treat C.difficile-associated disease (CDAD).

In one embodiment, the present antibodies or antigen-binding portionsthereof provide one or more of the following effects: protect from ortreat C. difficile-mediated colitis, antibiotic-associated colitis,pseudomembranous colitis (PMC) or other intestinal disease in a subject;protect from or treat diarrhea in a subject; and/or treat or inhibitrelapse of C. difficile-mediated disease. When administered to a mammal,the present antibodies or antigen-binding portions thereof protect themammal against toxin A administered in an amount that would be fatal tothe mammal had the antibody or antigen-binding portion thereof notadministered.

The present antibodies or antigen-binding portions thereof includeantibodies produced by hybridoma clone CAN20G2, CAN20G1, CAN20G5,CAN20G8, CAN19G1, CAN19G2 or CAN19G3 described herein.

Also encompassed by the present invention are antibodies orantigen-binding portions thereof that include an antigen-binding portionof an antibody produced by hybridoma clone CAN20G2, CAN20G1, CAN20G5,CAN20G8, CAN19G1, CAN19G2 or CAN19G3.

As used herein, CAN20G1, CAN20G2, CAN20G5, CAN20G8, CAN19G1, CAN19G2 andCAN19G3 refer to the hybridoma clones or the monoclonal antibodiesgenerated by the corresponding hybridoma clones.

The antibodies or antigen-binding portions thereof can specifically bindto an epitope within fragment 4 of toxin A, e.g., an epitope betweenamino acid residues 1853-2710 of toxin A. Babcock, G. J. et al.,Infection and Immunity, 74: 6339-6347 (2006). In other embodiments, theantibodies or antigen-binding portions thereof specifically bind to anepitope within fragment 1 (amino acid residues 1-659), fragment 2 (aminoacid residues 660-1256) or fragment 3 (amino acid residues 1257-1852) oftoxin A. In other embodiments, the antibodies or antigen-bindingportions thereof specifically bind an epitope within amino acid residues1-600, 400-600, 415-540, 1-100, 100-200, 200-300, 300-400, 400-500,500-600, 600-700, 700-800, 900-1000, 1100-1200, 1200-1300, 1300-1400,1400-1500, 1500-1600, 1600-1700, 1800-1900, 1900-200, 2100-2200 or2200-2300, 2300-2400, 2400-2500, 2500-2600, 2600-2710 of toxin A, or anyinterval, portion or range thereof.

The present antibodies, or antigen-binding portions thereof, include,but are not limited to, monoclonal antibodies, chimeric antibodies,humanized antibodies, polyclonal antibodies, recombinant antibodies, aswell as antigen-binding portions of the foregoing. An antigen-bindingportion of an antibody may include a portion of an antibody thatspecifically binds to a toxin of C. difficile (e.g., toxin A).

The humanized antibody of the present invention is an antibody from anon-human species where the amino acid sequence in the non-antigenbinding regions (and/or the antigen-binding regions) has been altered sothat the antibody more closely resembles a human antibody, and stillretains its original binding ability.

Humanized antibodies can be generated by replacing sequences of thevariable region that are not directly involved in antigen binding withequivalent sequences from human variable regions. Those methods includeisolating, manipulating, and expressing the nucleic acid sequences thatencode all or part of variable regions from at least one of a heavy orlight chain. Sources of such nucleic acid are well known to thoseskilled in the art and, for example, may be obtained from a hybridomaproducing an antibody against toxin A. The recombinant DNA encoding thehumanized antibody, or fragment thereof, can then be cloned into anappropriate expression vector.

An antibody light or heavy chain variable region consists of a frameworkregion interrupted by three hypervariable regions, referred to ascomplementarity determining regions (CDRs). In one embodiment, humanizedantibodies are antibody molecules from non-human species having one, twoor all CDRs from the non-human species and a framework region from ahuman immunoglobulin molecule.

The humanized antibodies of the present invention can be produced bymethods known in the art. For example, once non-human (e.g., murine)antibodies are obtained, variable regions can be sequenced, and thelocation of the CDRs and framework residues determined. Kabat, E. A., etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242. Chothia, C. et al. (1987) J. Mol. Biol., 196:901-917. Thelight and heavy chain variable regions can, optionally, be ligated tocorresponding constant regions. CDR-grafted antibody molecules can beproduced by CDR-grafting or CDR substitution. One, two, or all CDRs ofan immunoglobulin chain can be replaced. For example, all of the CDRs ofa particular antibody may be from at least a portion of a non-humananimal (e.g., mouse such as CDRs shown in Table 1) or only some of theCDRs may be replaced. It is only necessary to keep the CDRs required forbinding of the antibody to a predetermined antigen (e.g., toxin A of C.difficile). Morrison, S. L., 1985, Science, 229:1202-1207. Oi et al.,1986, BioTechniques, 4:214. U.S. Pat. Nos. 5,585,089; 5,225,539;5,693,761 and 5,693,762. EP 519596. Jones et al., 1986, Nature,321:552-525. Verhoeyan et al., 1988, Science, 239:1534. Beidler et al.,1988, J. Immunol., 141:4053-4060.

Also encompassed by the present invention are antibodies orantigen-binding portions thereof containing one, two, or all CDRs asdisclosed herein, with the other regions replaced by sequences from atleast one different species including, but not limited to, human,rabbits, sheep, dogs, cats, cows, horses, goats, pigs, monkeys, apes,gorillas, chimpanzees, ducks, geese, chickens, amphibians, reptiles andother animals.

A chimeric antibody is a molecule in which different portions arederived from different animal species. For example, an antibody maycontain a variable region derived from a murine mAb and a humanimmunoglobulin constant region. Chimeric antibodies can be produced byrecombinant DNA techniques. Morrison, et al., Proc Natl Acad Sci,81:6851-6855 (1984). For example, a gene encoding a murine (or otherspecies) monoclonal antibody molecule is digested with restrictionenzymes to remove the region encoding the murine Fc, and the equivalentportion of a gene encoding a human Fc constant region is substituted.Chimeric antibodies can also be created by recombinant DNA techniqueswhere DNA encoding murine V regions can be ligated to DNA encoding thehuman constant regions. Better et al., Science, 1988, 240:1041-1043. Liuet al. PNAS, 1987 84:3439-3443. Liu et al., J. Immunol., 1987,139:3521-3526. Sun et al. PNAS, 1987, 84:214-218. Nishimura et al.,Canc. Res., 1987, 47:999-1005. Wood et al. Nature, 1985, 314:446-449.Shaw et al., J. Natl. Cancer Inst., 1988, 80:1553-1559. InternationalPatent Publication Nos. WO1987002671 and WO 86/01533. European PatentApplication Nos. 184, 187; 171,496; 125,023; and 173,494. U.S. Pat. No.4,816,567.

The antibodies can be full-length or can include a fragment (orfragments) of the antibody having an antigen-binding portion, including,but not limited to, Fab, F(ab′)2, Fab′, F(ab)′, Fv, single chain Fv(scFv), bivalent scFv (bi-scFv), trivalent scFv (tri-scFv), Fd, dAbfragment (e.g., Ward et al., Nature, 341:544-546 (1989)), an isolatedCDR, diabodies, triabodies, tetrabodies, linear antibodies, single-chainantibody molecules, and multispecific antibodies formed from antibodyfragments. Single chain antibodies produced by joining antibodyfragments using recombinant methods, or a synthetic linker, are alsoencompassed by the present invention. Bird et al. Science, 1988,242:423-426. Huston et al., Proc. Natl. Acad. Sci. USA, 1988,85:5879-5883.

The antibodies or antigen-binding portions thereof of the presentinvention may be monospecific, bi-specific or multispecific.Multispecific or bi-specific antibodies or fragments thereof may bespecific for different epitopes of one target polypeptide (e.g., toxinA) or may contain antigen-binding domains specific for more than onetarget polypeptide (e.g., antigen-binding domains specific for toxin Aand toxin B; or antigen-binding domains specific for toxin A and otherantigen of C. difficile; or antigen-binding domains specific for toxin Aand other kind of bacterium or virus). In one embodiment, amultispecific antibody or antigen-binding portion thereof comprises atleast two different variable domains, wherein each variable domain iscapable of specifically binding to a separate antigen or to a differentepitope on the same antigen. Tuft et al., 1991, J. Immunol. 147:60-69.Kufer et al., 2004, Trends Biotechnol. 22:238-244. The presentantibodies can be linked to or co-expressed with another functionalmolecule, e.g., another peptide or protein. For example, an antibody orfragment thereof can be functionally linked (e.g., by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody or antibody fragmentto produce a bi-specific or a multispecific antibody with a secondbinding specificity. For example, the present invention includesbi-specific antibodies wherein one arm of an immunoglobulin is specificfor toxin A, and the other arm of the immunoglobulin is specific for asecond therapeutic target or is conjugated to a therapeutic moiety suchas a trypsin inhibitor.

All antibody isotypes are encompassed by the present invention,including IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), IgDor IgE. The antibodies or antigen-binding portions thereof may bemammalian (e.g., mouse, human) antibodies or antigen-binding portionsthereof. The light chains of the antibody may be of kappa or lambdatype.

The CDRs of the present antibodies or antigen-binding portions thereofcan be from a non-human or human source. The framework of the presentantibodies or antigen-binding portions thereof can be human, humanized,non-human (e.g., a murine framework modified to decrease antigenicity inhumans), or a synthetic framework (e.g., a consensus sequence).

In one embodiment, the present antibodies, or antigen-binding portionsthereof, contain at least one heavy chain variable region and/or atleast one light chain variable region. The heavy chain variable region(or light chain variable region) contains three CDRs and four frameworkregions (FRs), arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Kabat, E. A., etal. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242,1991. Chothia, C. et al., J. Mol. Biol. 196:901-917, 1987.

The present antibodies or antigen-binding portions thereof specificallybind to toxin A with a dissociation constant (K_(D)) of less than about10⁻⁷ M, less than about 10⁻⁸ M, less than about 10⁻⁹ M, less than about10⁻¹⁰ M, less than about 10⁻¹¹ M, or less than about 10⁻¹² M.

Antibodies with a variable heavy chain region and a variable light chainregion that are at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, atleast about 99%, about 70%, about 75%, about 80%, about 81%, about 82%,about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, about 99% or about 100% homologous tothe variable heavy chain region and variable light chain region of theantibody produced by clone CAN20G1, CAN20G2, CAN20G5, CAN20G8, CAN19G1,CAN19G2 or CAN19G3 can also bind to toxin A.

In related embodiments, anti-toxin A antibodies or antigen-bindingportions thereof include, for example, the CDRs of variable heavy chainsand/or variable light chains of CAN20G1, CAN20G2, CAN20G5, CAN20G8,CAN19G1, CAN19G2 or CAN19G3. The CDRs of the variable heavy chainregions from these clones, as well as the CDRs of the variable lightchain regions from these clones, are shown in Table 1.

TABLE 1 Seq ID Nos. 3-104 Chain, Seq ID Name Region Sequence No.Fragment GWQTINGKKYYFDINTGAALISYKIINGKHFYFNNDG   3 4 of ToxinVMQLGVFKGPDGFEYFAPANTQNNNIEGQAIVYQSK AFLTLNGKKYYFDNDSKAVTGWRIINNEKYYFNPNNAIAAVGLQVIDNNKYYFNPDTAIISKGWQTVNGSRYYFDTDTAIAFNGYKTIDGKHFYFDSDCVVKIGVFSTSNGFEYFAPANTYNNNIEGQAIVYQSKFLTLNGKKYYFD NNSKAVTGWQTIDSKKYYFNTNTAEAATGWQTIDGKKYYFNTNTAEAATGWQTIDGKKYYFNTNTAIASTGYTIINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTDANNIEGQAILYQNEFLTLNGKKYYFGSDSKAVTGWRIINNKKYYFNPNNAIAAIHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVT GWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNLNTAEAATGWQTIDGKKYYFNTNTFIASTGYTSINGKHFYFNTDGIMQIGVFKGPNGFEYFAPANTHNNNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFNTNTSIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNNSKAATGWVTIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGSNGFEYFAPANTDANNIEGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINGKVYYFMPDTAMAAAGGLF EIDGVIYFFGVDGVKAPGIYG CAN20G1 K,QVVLTQSPAIMSASLGERVTMTCTASSSVISSYLHWY   4 variableQQKPGSSPKLWIYSTSTLASGVPARFSGSGSGTSYSLT regionISSMEAEDAATYYCLQYHRSPRTFGGGTKLEIK CAN20G 1 K, SSVISSY   5 CDR1 CAN20G1K, STS   6 CDR2 CAN20G1 K, CLQYHRSPRTF   7 CDR3 CAN20G1 K,QVVLTQSPAIMSASLGERVTMTCTAS   8 FR1 CAN20G1 K, LHWYQQKPGSSPKLWIY   9 FR2CAN20G1 K, TLASGVPARFSGSGSGTSYSLTISSMEAEDAATYY  10 FR3 CAN20G1 K,GGGTKLEIK  11 FR4 CAN20G1 H, QIQLVQSGPELKKPGETVKISCKASGYTFTNDGMNW  12variable VKQAPGKGLKWMGWINTNTGEPTYVEEFKGRFAFS regionLETSASTAYLQINNLKNEDTATYFCYVNYDYYTMDC WGQGTSVTVSS CAN20G1 H, GYTFTNDG  13CDR1 CAN20G1 H, INTNTGEP  14 CDR2 CAN20G1 H, CYVNYDYYTMDCW  15 CDR3CAN20G1 H, QIQLVQSGPELKKPGETVKISCKAS  16 FR1 CAN20G1 H,MNWVKQAPGKGLKWMGW  17 FR2 CAN20G1 H,TYVEEFKGRFAFSLETSASTAYLQINNLKNEDTATYF  18 FR3 CAN20G1 H, GQGTSVTVSS  19FR4 CAN20G2 K, QVVLTQSPAIMSASLGDRVTMTCTASSSVISTYLHWY  20 variableQQKPGSSPKLWIYSTSTLASGVPPRFSGSGSGTSYSLT regionISSMEAEDAATYYCLQYHRSPRTFGGGTKLEIK CAN20G2 K, SSVISTY  21 CDR1 CAN20G2 K,STS  22 CDR2 CAN20G2 K, LQYHRSPRT  23 CDR3 CAN20G2 K,QVVLTQSPAIMSASLGDRVTMTCTAS  24 FR1 CAN20G2 K, LHWYQQKPGSSPKLWIY  25 FR2CAN20G2 K, TLASGVPPRFSGSGSGTSYSLTISSMEAEDAATYYC  26 FR3 CAN20G2 K,FGGGTKLEIK  27 FR4 CAN20G2 H, QIQLVQSGPEVKKPGETVKISCKASGYTFTNQGMNW  28variable VKQAPGKGLKWMGWINTNTGEPTYTEEFKGRFAFSL regionETSASTAYLQINNLKNEDTATYFCYVNYDYYTMDF WGQGTSVTVSS CAN20G2 H, GYTFTNQG  29CDR1 CAN20G2 H, INTNTGEP  30 CDR2 CAN20G2 H, YVNYDYYTMDF  31 CDR3CAN20G2 H, QIQLVQSGPEVKKPGETVKISCKAS  32 FR1 CAN20G2 H,MNWVKQAPGKGLKWMGW  33 FR2 CAN20G2 H,TYTEEFKGRFAFSLETSASTAYLQINNLKNEDTATYF  34 FR3 C CAN20G2 H, WGQGTSVTVSS 35 FR4 CAN20G5 K, QIVLTQSPAIMSASLGERVTMTCTASSSVYSTYLHWY  36 variableQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSL regionTISSMEAEDAATYYCHQYHRSPRTFGGGTKLEIK CAN20G5 K, SSVYSTY  37 CDR1 CAN20G5K, STS  38 CDR2 CAN20G5 K, CHQYHRSPRTF  39 CDR3 CAN20G5 K,QIVLTQSPAIMSASLGERVTMTCTAS  40 FR1 CAN20G5 K, LHWYQQKPGSSPKLWIY  41 FR2CAN20G5 K, NLASGVPARFSGSGSGTSYSLTISSMEAEDAATYY  42 FR3 CAN20G5 K,GGGTKLEIK  43 FR4 CAN20G5 H, QIQLVQSGPELKKPGETVKISCKASGYSFTNSGMNW  44variable VKEAPGKGLKWMGWINTNTGEPTYAEEFMGRFAFS regionLETSASTAYLQINNLKNEDTATYFCYVNYDYYTIDY WGQGTSVTVSS CAN20G5 H, GYSFTNSG  45CDR1 CAN20G5 H, INTNTGEP  46 CDR2 CAN20G5 H, CYVNYDYYTIDYW  47 CDR3CAN20G5 H, QIQLVQSGPELKKPGETVKISCKAS  48 FR1 CAN20G5 H,MNWVKEAPGKGLKWMGW  49 FR2 CAN20G5 H,TYAEEFMGRFAFSLETSASTAYLQINNLKNEDTATYF  50 FR3 CAN20G5 H, GQGTSVTVSS  51FR4 CAN20G8 K, QVVLTQSPAIMSASLGERVTMTCTASSSVISSYLHWY  52 variableQQKPGSSPKLWIYSTSILASGVPARFSGSGSGTSYSLTI regionSSMEAEDAATYYCLQYHRSPRTFGGGTKLEIK CAN20G8 K, SSVISSY  53 CDR1 CAN20G8 K,STS  54 CDR2 CAN20G8 K, CLQYHRSPRTF  55 CDR3 CAN20G8 K,QVVLTQSPAIMSASLGERVTMTCTAS  56 FR1 CAN20G8 K, LHWYQQKPGSSPKLWIY  57 FR2CAN20G8 K, ILASGVPARFSGSGSGTSYSLTISSMEAEDAATYY  58 FR3 CAN20G8 K,GGGTKLEIK  59 FR4 CAN20G8 H, QIQLVQSGPELKKPGETVKISCKASGYAFTNDGMNW  60variable VKQAPGKGLKWMGWINTNTGEPTYAEEFKGRFAFS regionLETSASTAYLQINNLKNEDTATYFCYVNYDYYTMDC WGQGTSVTVSS CAN20G8 H, GYAFTNDG  61CDR1 CAN20G8 H, INTNTGEP  62 CDR2 CAN20G8 H, CYVNYDYYTMDCW  63 CDR3CAN20G8 H, QIQLVQSGPELKKPGETVKISCKAS  64 FR1 CAN20G8 H,MNWVKQAPGKGLKWMGW  65 FR2 CAN20G8 H,TYAEEFKGRFAFSLETSASTAYLQINNLKNEDTATYF  66 FR3 CAN20G8 H, GQGTSVTVSS  67FR4 CAN20G1 Caagttgttctcacccagtctccagcaatcatgtctgcatctctaggggaacgggtca 68 Kappa ccatgacctgcactgccagctcaagtgtaatttccagttatttgcactggtaccagcagaagccaggatcctcccccaaactctggatttatagcacatccaccctggcttctggagtcccagctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgcctccagtatcatcgttccccacggacgttcggtggaggcaccaagctggaaatcaaacgggctgatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggcgtcctgaacagttggactgatcaggacagcaaagacagcac aag CAN20G1Cagatccagttggtgcagtctggacctgagctgaagaagcctggagagacagtca  69 Heavyagatctcctgcaaggcttctgggtataccttcacaaacgatggaatgaactgggtgaaacaggctccaggaaagggtttaaagtggatgggctggataaacaccaacactggagagccaacatatgttgaagagttcaagggacggtttgccttctctttagaaacctctgccagcactgcctatttgcagatcaacaacctcaaaaatgaggacacggctacatatttctgttatgttaactacgattattatactatggactgctggggtcaaggaacctcagtcaccgtctcctcagccaaaacgacacccccatctgtctatccactggcccctggatctgctgcccaaactaactccatggtgaccctgggatgcctggtcaagggctatttccctgagccagtgacagtgacctggaactctggatccctgtccagcggtgtgcacaccttccca gctstcctaagCAN20G2 Caagttgttctcacccagtctccagcaatcatgtctgcatctctaggggatcgggtca  70Kappa ccatgacctgcactgccagctcaagtgtaatttccacttacttgcactggtatcagcagaagccaggatcctcccccaaactctggatttatagcacatccaccctggcttctggagtcccacctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgcctccagtatcaccgttccccacggacgttcggtggaggcaccaagctggaaatcaaacgggctgatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggcgtcctgaacagttggactgatcaggacagcaaagacagcac aag CAN20G2Cagatccagttggtgcagtctggacctgaggtgaagaagcctggagagacagtca  71 Heavyagatctcctgcaaggcttctgggtataccttcacaaaccaaggaatgaactgggtgaaacaggctccaggaaagggtttaaagtggatgggctggataaacaccaacactggagagccaacatatactgaagagttcaagggacggtttgccttctctttagaaacctctgccagcactgcctatttgcagatcaacaacctcaaaaatgaggacacggctacatatttctgttatgttaactacgattattatactatggacttctggggtcaaggaacctcggtcaccgtctcctcagccaaaacaacagccccatcggtctatccactggcccctgtgtgtggagatacaactggctcctcggtgactctaggatgcctggtcaagggttatttccctgagccagtgaccttgacctggaactctggatccctgtccagtggtgtgcacaccttccca gctstcctaagCAN20G5 Caaattgttctcacccagtctccagcaatcatgtctgcttctctaggggaacgggtca  72Kappa ccatgacctgcactgccagctcaagtgtatattccacttacttgcactggtaccagcagaagccaggatcctcccccaaactctggatttatagcacatccaacctggcttctggagtcccagctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccaccagtatcatcgttccccacggacgttcggtggaggcaccaagctggaaatcaaacgggctgatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggcgtcctgaacagttggactgatcaggacagcaaagacagc acaag CAN20G5Cagatccagttggtacagtctggacctgagctgaagaagcctggagagacagtca  73 Heavyagatctcctgcaaggcttctgggtattccttcacaaactctggaatgaactgggtgaaagaggctccaggaaagggtttaaagtggatgggctggataaacaccaacactggagagccaacatatgctgaagaattcatgggacggtttgccttctctttggaaacctctgccagcactgcctatttgcagatcaacaacctcaaaaatgaagacacggctacatatttctgttatgttaactacgattactatactatagactactggggtcaaggaacctcagtcaccgtctcctcagccaaaacgacacccccatctgtctatccactggcccctggatctgctgcccaaactaactccatggtgaccctgggatgcctggtcaagggctatttccctgagccagtgacagtgacctggaactctggatccctgtccagcggtgtgcacaccttccca gctstcctaagCAN20G8 Cactggtaccagcagaagccaggatcctcccccaaactctggatttatagcacatc  74Kappa catcctggcttctggagtcccagctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgcctccagtatcatcgttccccacggacgttcggtggaggcaccaagctggaaatcaaacgggctgatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggcgtcctgaacagttggactgatcaggacagcaaagacagcacaag CAN20G8Cagatccagttggtgcagtctggacctgagctgaagaagcctggagagacagtca  75 Heavyagatctcctgcaaggcttctgggtatgccttcacaaacgatggaatgaactgggtgaaacaggctccaggaaagggtttaaagtggatgggctggataaacaccaacactggagagccaacatatgctgaagagttcaagggacggtttgccttctctttagaaacctctgccagcactgcctatttgcagatcaacaacctcaaaaatgaggacacggctacatatttctgttatgttaactacgattattatactatggactgctggggtcaaggaacctcagtcaccgtctcctcagccaaaacgacacccccatctgtctatccactggcccctggatctgctgcccaaactaactccatggtgaccctgggatgcctggtcaagggctatttccctgagccagtgacagtgacctggaactctggatccctgtccagcggtgtgcacaccttc ccagctstcctaag5′mVK- GGTGCAGATTTTCAGCTTCC  76 Lead-1 3′Kappa GTGCTGTCTTTGCTGTCCTG  77ConstRT 5′mVH- BTNCTYYTCTKCCTGRT  78 Lead-2 5′mVH- TGGSTGTGGAMCTTGCTATT 79 Lead-2A 3′mIG1- AGGASAGCTGGGAAGGTGTG  80 2C RT 5′mVK-CTWKGRSTKCTGCTKYTCTG  81 Lead-3 5′mVK- CCTGTTAGGCTGTTGGTGCT  82 Lead-3A5′mVH- RKCARCARCTRCAGGTGTCC  83 IGHV1- Lead 5′mVH-CCYWNTTTTAMAWGGTGTCCAKTGT  84 Lead-1 5′mVH- GGATGGAGCTRTATCATBCTC  85Lead-3 5′mVH- GRTCTTTMTYTTHHTCCTGTCA  86 Lead-4 5′mVH-VCCTTWMMTGGTATCCWGTST  87 Lead-5 CDR- H,GCCGCCACCATGGCATGCCCTGGCTTCCTGTGGGC  88 huCAN20 variableACTTGTGATCTCCACCTGTCTTGAATTTTCCATGGC G2 regionTCaggtgcagctggtgcaatctgggtctgagttgaagaagcctggggcctcagtg (FIG. 13A)aaggtttcctgcaaggcttctGGGTATACCTTCACAAACCAAGGAAtgaattgggtgcgacaggcccctggacaagggcttgagtggatgggatggATAAACACCAACACTGGAGAGCCAAcgtatgcccagggcttcacaggacggtttgtcttctccttggacacctctgtcagcacggcatatctgcagatcagcagcctaaaggctgaggacactgccgtgtattactgtTATgtcaatTACGATTATTATACTATGGACTTCtgggggcaagggaccacggtcaccgt ctcctca CDR- H,QVQLVQSGSELKKPGASVKVSCKASGYTFTNQGMN  89 huCAN20G2 variableWVRQAPGQGLEWMGWINTNTGEPTYAQGFTGRFVF (FIG. 13A) regionSLDTSVSTAYLQISSLKAEDTAVYYCYVNYDYYTMD FWGQGTTVTVSS CDR- K,GCCGCCACCATGGCATGCCCTGGCTTCCTGTGGGC  90 huCAN20G2 variableACTTGTGATCTCCACCTGTCTTGAATTTTCCATGGC (FIG. 13B) regionTGacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtTCAAGTGTAATTTCCACTTACTtaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatAGCACATCCAgtttgcaaagtggggteccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtCTCCAGTATCACCGTTCCCCACGGACGttcggcggaggga ccaaggtggagatcaaa CDR- K,DIQMTQSPSSLSASVGDRVTITCRASSSVISTYLNWYQ  91 huCAN20G2 variableQKPGKAPKLLIYSTSSLQSGVPSRFSGSGSGTDFTLTIS (FIG. 13B) regionSLQPEDFATYYCLQYHRSPRTFGGGTKVEIK HE- H,GCCGCCACCATGGCATGCCCTGGCTTCCTGTGGGC  92 huCAN20G2 variableACTTGTGATCTCCACCTGTCTTGAATTTTCCATGGC (FIG. 14A) regionTCAGatcCAGttgGTGcagTCTggaCCTgag CTG aagAAGcctGGAgagACAgtcAAGatcTCCtgcAAGgctTCTgggTATaccTTCacaAACcaaGGAatgAACtggGTGaaaCAGgctCCAggaAAGggtTTAaagTGGatgGGCtggATAaacACCaacACTggaG AGccaACAtatACT GCCGAT ttc ACAggaCGGtttGCCttcTC TttaGAAaccTCT GTG AGCactGCCtatTTGcagATCaac TC C ctcAAAGCT GAGgacACGgctACAtatTTCtgtTATgtcaattacGATtatTATactATGgacTTCTGGGGTCAAGGAacc CTG gtcACCgtcTCCtca HE- H,QIQLVQSGPE L KKPGETVKISCKASGYTFTNQGMNW  93 huCAN20G2 variableVKQAPGKGLKWMGWINTNTGEPTYT AD F T GRFAFS (FIG. 14A) region LETS VSTAYLQIN S LK A EDTATYFCYVNYDYYTMDF WGQGT L VTVSS HE- K,GCCGCCACCATGGCATGCCCTGGCTTCCTGTGGGC  94 huCAN20G2 variableACTTGTGATCTCCACCTGTCTTGAATTTTCCATGGC (FIG. 14B) region T GAC gtt CAGctcACCcagTCTcca AGC atcATGtctGCAtctCTAgggGATcggGTCaccATGaccTGCactGCCagcTCAagtGTAattTCCactTACttgCACtggTATcagCAGaagCCAggaTCCtccCCCaaaCTCtggATTtatAGCacaTCCaccCTGgctTCTggaG TCcca AGCcgcTTCagtGGCagtGGGtctGGGacc GAC tacTC TctcACAatcAGCagcATGgag CCTgaaGATgctGCCactTAT tacTGCctcCAGtatCACcgtTCCccaCGGacgTTCggtGGAgg cACCaagGTG gaaATCaaa HE- K, D V Q LTQSP S IMSASLGDRVTMTCTASSSVISTYLHWY  95huCAN20G2 variable QQKPGSSPKLWIYSTSTLASGVPPRFSGSGSGT D YSLT (FIG. 14B)region ISSME P EDAATYYCLQYHRSPRTFGGGTK V EIK HE- HGCCGCCACCATGGCATGCCCTGGCTTCCTGTGGGC  96 huCAN20G2ACTTGTGATCTCCACCTGTCTTGAATTTTCCATGGC (FIG. 15)TCAGatcCAGttgGTGcagTCTggaCCTgag CTG aagAAGcctGGAgagACAgtcAAGatcTCCtgcAAGgctTCTgggTATaccTTCacaAACcaaGGAatgAACtggGTGaaaCAGgctCCAggaAAGggtTTAaagTGGatgGGCtggATAaacACCaacACTggaG AGccaACAtatACT GCCGAT ttc ACAggaCGGtttGCCttcTC TttaGAAaccTCT GTG AGCactGCCtatTTGcagATCaac TC C ctcAAAGCT GAGgacACGgctACAtatTTCtgtTATgtcaattacGATtatTATactATGgacTTCTGGGGTCAAGGAacc CTG gtcACCgtcTCCtcaGGTGAGTGCGGCCGCGAGCCCAG ACACTGGACGCTGAACCTCGCGGACAGTTAAGAACCCAGGGGCCTCTGCGCCCTGGGCCCAGCTCTGTCC CACACCGCGGTCACATGGCACCACCTCTCTTGCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGG CACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCC CCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGC ACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTG GT GAGAGGCCAGCACAGGGAGGGAGGGTGTCTGCTGGAAGCCAGGCTCAGCGCTCCTGCCTGGACGCATCC CGGCTATGCAGTCCCAGTCCAGGGCAGCAAGGCAGGCCCCGTCTGCCTCTTCACCCGGAGGCCTCTGCCC GCCCCACTCATGCTCAGGGAGAGGGTCTTCTGGCTTTTTCCCCAGGCTCTGGGCAGGCACGGGCTAGGTG CCCCTAACCCAGGCCCTGCACACAAAGGGGCAGGTGCTGGGCTCAGACCTGCCAAGAGCCATATCCGGGA GGACCCTGCCCCTGACCTAAGCCCACCCCAAAGGCCAAACTCTCCACTCCCTCAGCTCGGACACCTTCTCTCCTCCCAGATTCCAGTAACTCCCAATCTTCTCTCTG CAG AGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAG GTAAGCCAGCCCAGGCCTC GCCCTCCAGCTCAAGGCGGGACAGGTGCCCTAGAGTAGCCTGCATCCAGGGACAGGCCCCAGCCGGGTGC TGACACGTCCACCTCCATCTCTTCCTCAG CACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC CCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGAC GTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC CATCGAGAAAACCATCTCCAAAGCCAAAG GTGGGACCCGTGGGGTGCGAGGGCCACATGGACAGAGG CCGGCTCGGCCCACCCTCTGCCCTGAGAGTGACCGCTGTACCAACCTCTGTCCCTACAG GGCAGCCCCG AGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGA CCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCG GAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAG CTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGC TCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGATGAGCTAGC HE- H QIQLVQSGPELKKPGETVKISCKASGYTFTNQGMNW 97 huCAN20G2 VKQAPGKGLKWMGWINTNTGEPTYTADFTGRFAFS (FIG. 15)LETSVSTAYLQINSLKAEDTATYFCYVNYDYYTMDFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK HE- KGCCGCCACCATGGCATGCCCTGGCTTCCTGTGGGC  98 huCAN20G2ACTTGTGATCTCCACCTGTCTTGAATTTTCCATGGC (FIG. 16) T GAC gtt CAGctcACCcagTCTcca AGC atcATGtctGCAtctCTAgggGATcggGTCaccATGaccTGCactGCCagcTCAagtGTAattTCCactTACttgCACtggTATcagCAGaagCCAggcAGCtccCCCaaaCTCtggATTtatAGCacaTCCaccCTGgctTCTgga GTCcca AGCcgcTTCagtGGCagtGGGtctGGGacc GAC tacT CTctcACAatcAGCagcATGgag CCTgaaGATgctGCCactTA TtacTGCctcCAGtatCACcgtTCCccaCGGacgTTCggtGGAg gcACCaagGTG gaaATCaaaC GTAAGTGCACTTTGCGG CCGCTAGGAAGAAACTCAAAACATCAAGATTTTAAATACGCTTCTTGGTCTCCTTGCTATAATTATCTGGGATAAGCATGCTGTTTTCTGTCTGTCCCTAACATGCC CTGTGATTATCCGCAAACAACACACCCAAGGGCAGAACTTTGTTACTTAAACACCATCCTGTTTGCTTCTT TCCTCAG GAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTG GAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGG TGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCC TGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA TAGTTAACG HE- KDVQLTQSPSIMSASLGDRVTMTCTASSSVISTYLHWY  99 huCAN20G2QQKPGSSPKLWIYSTSTLASGVPPRFSGSGSGTDYSLT (FIG. 16)ISSMEPEDAATYYCLQYHRSPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC AVA-K GCCGCCACCATGGCATGCCCTGGCTTCCTGTGGGC 100 huCAN20G2ACTTGTGATCTCCACCTGTCTTGAATTTTCCATGGC (FIG. 18)TGACatcCAGatgACCcagTCTccaTCCtccCTGtctGCAtctG TAggaGACagaGTCaccATCactTGCAGC GCGagtTCAAG TGTAATTTCCACTTACTTAaatTGGtatCAGcagAAAcca GGGaaaGCCcctAAGgtg CTGatcTACAGCACATCC AGC ttgcacagcGGGgtcCCAtcaAGGttcAGTggaAGTggaTCTggg ACAgatTTTact ctgaccATCagcAGCctgCAGcctGAAgat ttc gcaACAtatTACtgtCTCCAGTATCACCGTTCCCCACGGACGttcggccaagggaccaaggtggaaatcaaaC GTAAGTGCACTTTGCGGCCGCTAGGAAGAAACTCAAAACATCAAGAT TTTAAATACGCTTCTTGGTCTCCTTGCTATAATTATCTGGGATAAGCATGCTGTTTTCTGTCTGTCCCTAAC ATGCCCTGTGATTATCCGCAAACAACACACCCAAGGGCAGAACTTTGTTACTTAAACACCATCCTGTTTGC TTCTTTCCTCAG GAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTG GAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGG ACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCT ACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT GTTGATAGTTAACG Chimeric HGCCGCCACCATGGCATGCCCTGGCTTCCTGTGGGC 101 CAN20G2ACTTGTGATCTCCACCTGTCTTGAATTTTCCATGGC (FIG. 19A)TCAGATCCAGTTGGTGCAGTCTGGACCTGAGGTGA AGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAAACCAAGGAATGA ACTGGGTGAAACAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCAACACTGGAGAG CCAACATATACTGAAGAGTTCAAGGGACGGTTTGCCTTCTCTTTAGAAACCTCTGCCAGCACTGCCTATTT GCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTTATGTTAACTACGATTATTATACT ATGGACTTCTGGGGTCAAGGAACCTCGGTCACCGTCTCCTCAG GTGAGTGCGGCCGCGAGCCCAGACACT GGACGCTGAACCTCGCGGACAGTTAAGAACCCAGGGGCCTCTGCGCCCTGGGCCCAGCTCTGTCCCACA CCGCGGTCACATGGCACCACCTCTCTTGCAG CCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACC CTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGA ACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCA GACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTG GTGAGAG GCCAGCACAGGGAGGGAGGGTGTCTGCTGGAAGCCAGGCTCAGCGCTCCTGCCTGGACGCATCCCGGCT ATGCAGTCCCAGTCCAGGGCAGCAAGGCAGGCCCCGTCTGCCTCTTCACCCGGAGGCCTCTGCCCGCCCC ACTCATGCTCAGGGAGAGGGTCTTCTGGCTTTTTCCCCAGGCTCTGGGCAGGCACGGGCTAGGTGCCCCTA ACCCAGGCCCTGCACACAAAGGGGCAGGTGCTGGGCTCAGACCTGCCAAGAGCCATATCCGGGAGGACC CTGCCCCTGACCTAAGCCCACCCCAAAGGCCAAACTCTCCACTCCCTCAGCTCGGACACCTTCTCTCCTCC CAGATTCCAGTAACTCCCAATCTTCTCTCTGCAGA GCCCAAATCTTGTGACAAAACTCACACATGCCC ACCGTGCCCAG GTAAGCCAGCCCAGGCCTCGCCCTCCAGCTCAAGGCGGGACAGGTGCCCTAGAGTAGC CTGCATCCAGGGACAGGCCCCAGCCGGGTGCTGACACGTCCACCTCCATCTCTTCCTCAG CACCTGAACT CCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGAC CCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTA CGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACG TACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC AAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAG GTGGGACC CGTGGGGTGCGAGGGCCACATGGACAGAGGCCGGCTCGGCCCACCCTCTGCCCTGAGAGTGACCGCTGT ACCAACCTCTGTCCCTACAG GGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAG GAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTC CGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACG TCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT CTCCGGGTAAATGATGA Chimeric HAATMACPGFLWALVISTCLEFSMAQIQLVQSGPEVK 102 CAN20G2KPGETVKISCKASGYTFTNQGMNWVKQAPGKGLKW (FIG. 19A)MGWINTNTGEPTYTEEFKGRFAFSLETSASTAYLQINNLKNEDTATYFCYVNYDYYTMDFWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK Chimeric KGCCGCCACCATGGCATGCCCTGGCTTCCTGTGGGC 103 CAN20G2ACTTGTGATCTCCACCTGTCTTGAATTTTCCATGGC (FIG.19B)TCAAGTTGTTCTCACCCAGTCTCCAGCAATCATGTC TGCATCTCTAGGGGATCGGGTCACCATGACCTGCACTGCCAGCTCAAGTGTAATTTCCACTTACTTGCACTGGTATCAGCAGAAGCCAGGcTCtTCCCCCAAACTCT GGATTTATAGCACATCCACCCTGGCTTCTGGAGTCCCACCTCGCTTCAGTGGCAGTGGGTCTGGGACCTC TTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCTCCAGTATCACCGTT CCCCACGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC GTAAGTGCACTTTGCGGCCGCTAGGAAG AAACTCAAAACATCAAGATTTTAAATACGCTTCTTGGTCTCCTTGCTATAATTATCTGGGATAAGCATGCTGTTTTCTGTCTGTCCCTAACATGCCCTGTGATTATC CGCAAACAACACACCCAAGGGCAGAACTTTGTTACTTAAACACCATCCTGTTTGCTTCTTTCCTCAG GAAC TGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTC TGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACG CCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGC CTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTC ACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGATAG Chimeric KAATMACPGFLWALVISTCLEFSMAQVVLTQSPAIMS 104 CAN20G2ASLGDRVTMTCTASSSVISTYLHWYQQKPGSSPKLWI (FIG.19B)YSTSTLASGVPPRFSGSGSGTSYSLTISSMEAEDAATYYCLQYHRSPRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECIn Table 1, the CDRs are IMGT numbering. H: heavy chain; K: kappa chain.

In certain embodiments, the antibodies or antigen-binding portionsthereof include a variable heavy chain region comprising an amino acidsequence at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%,about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99% or about 100% homologous to a variableheavy chain region amino acid sequence of the antibody produced by cloneCAN20G1 (SEQ ID NO: 12), CAN20G2 (SEQ ID NO: 28), CAN20G5 (SEQ ID NO:44), or CAN20G8 (SEQ ID NO: 60).

In certain embodiments, the antibodies or antigen-binding portionsthereof include a variable light chain region comprising an amino acidsequence at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about99%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%,about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99% or about 100% homologous to a variablelight chain region amino acid sequence of the antibody produced by cloneCAN20G1 (SEQ ID NO: 4), CAN20G2 (SEQ ID NO: 20), CAN20G5 (SEQ ID NO:36), or CAN20G8 (SEQ ID NO: 52).

In certain embodiments, the antibodies or antigen-binding portionsthereof each include both a variable heavy chain region comprising anamino acid sequence at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 99%, about 70%, about 75%, about 80%, about 81%, about82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 99% or about 100% homologousto a variable heavy chain region amino acid sequence of the antibodyproduced by clone CAN20G1 (SEQ ID NO: 12), CAN20G2 (SEQ ID NO: 28),CAN20G5 (SEQ ID NO: 44), or CAN20G8 (SEQ ID NO: 60), and a variablelight chain region including an amino acid sequence at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 99%, about 70%, about 75%,about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about99% or about 100% homologous to a variable light chain amino acidsequence of clone CAN20G1 (SEQ ID NO: 4), CAN20G2 (SEQ ID NO: 20),CAN20G5 (SEQ ID NO: 36), or CAN20G8 (SEQ ID NO: 52).

In various embodiments, the antibodies or antigen-binding portionsthereof specifically bind to an epitope that overlaps with, or are atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 99%, about70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%,about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99% or about 100% homologous to, an epitope bound by anantibody produced by clone CAN20G1, CAN20G2, CAN20G5, or CAN20G8 and/orcompete for binding to toxin A with an antibody produced by cloneCAN20G1, CAN20G2, CAN20G5, or CAN20G8.

A variable heavy chain region of the antibodies or antigen-bindingportions thereof can comprise one, two three or more complementaritydetermining regions (CDRs) that are at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 99%, about 70%, about 75%, about 80%,about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100% homologous to CDRs of the antibody produced by clone CAN20G1(SEQ ID NOs: 13, 14, 15), CAN20G2 (SEQ ID NOs: 29, 30, 31), CAN20G5 (SEQID NOs: 45, 46, 47), or CAN20G8 (SEQ ID NOs: 61, 62, 63).

A variable light chain region of the antibodies or antigen-bindingportions thereof can comprise one, two three or more CDRs that are atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 99%, about70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%,about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99% or about 100% homologous to CDRs of a variablelight chain region of the antibody produced by clone CAN20G1 (SEQ IDNOs: 5, 6, 7), CAN20G2 (SEQ ID NOs: 21, 22, 23), CAN20G5 (SEQ ID NOs:37, 38, 39), or CAN20G8 (SEQ ID NOs: 53, 54, 55).

A variable heavy chain region of the antibodies or antigen-bindingportions thereof can comprise one, two three or more complementaritydetermining regions (CDRs) that are at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 99%, about 70%, about 75%, about 80%,about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100% homologous to CDRs of the antibody produced by clone CAN20G1(SEQ ID NOs: 13-15), CAN20G2 (SEQ ID NOs: 29-31), CAN20G5 (SEQ ID NOs:45-47), or CAN20G8 (SEQ ID NOs: 61-63), and a variable light chainregion of the antibodies or antigen-binding portions thereof cancomprise one, two three or more CDRs that are at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 99%, about 70%, about 75%, about80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%,about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100% homologous to CDRs of a variable light chain region of theantibody produced by clone CAN20G1 (SEQ ID NOs: 5-7), CAN20G2 (SEQ IDNOs: 21-23), CAN20G5 (SEQ ID NOs: 37-39), or CAN20G8 (SEQ ID NOs:53-55).

A variable heavy chain region of the antibodies or antigen-bindingportions thereof can include three CDRs that are at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 99%, about 70%, about 75%, about80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%,about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100% homologous to CDRs of a variable heavy chain region of theantibody produced by clone CAN20G1 (SEQ ID NOs: 13-15), CAN20G2 (SEQ IDNOs: 29-31), CAN20G5 (SEQ ID NOs: 45-47), or CAN20G8 (SEQ ID NOs:61-63).

In one embodiment, a variable light chain region of the antibodies orantigen-binding portions thereof includes three CDRs that are at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 99%, about 70%,about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, about 99% or about 100% homologous to CDRs of a variable lightchain region of the antibody produced by CAN20G1 (SEQ ID NOs: 5-7),CAN20G2 (SEQ ID NOs: 21-23), CAN20G5 (SEQ ID NOs: 37-39), or CAN20G8(SEQ ID NOs: 53-55).

In one embodiment, a variable heavy chain region of the antibodies orantigen-binding portions thereof includes three CDRs that are at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, at least about 99%, about 70%,about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, about 99% or about 100% homologous to CDRs of a variable heavychain region of the antibody produced by clone CAN20G1 (SEQ ID NOs:13-15), CAN20G2 (SEQ ID NOs: 29-31), CAN20G5 (SEQ ID NOs: 45-47), orCAN20G8 (SEQ ID NOs: 61-63), and a variable light chain region of theantibodies or antigen-binding portions thereof includes one, two orthree CDRs that are at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 99%, about 70%, about 75%, about 80%, about 81%, about82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 99% or about 100% homologousto CDRs of a variable light chain region of the antibody produced byclone CAN20G1 (SEQ ID NOs: 5-7), CAN20G2 (SEQ ID NOs: 21-23), CAN20G5(SEQ ID NOs: 37-39), or CAN20G8 (SEQ ID NOs: 53-55).

In certain embodiments, a variable heavy chain region of the antibodiesor antigen-binding portions thereof includes three CDRs that arehomologous to CDRs of a variable heavy chain region of the antibodyproduced by clone CAN20G1 (SEQ ID NOs: 13-15), CAN20G2 (SEQ ID NOs:29-31), CAN20G5 (SEQ ID NOs: 45-47), or CAN20G8 (SEQ ID NOs: 61-63), anda variable light chain region of the antibodies or antigen-bindingportions thereof includes three CDRs that are homologous to CDRs of avariable light chain region of the antibody produced by clone CAN20G1(SEQ ID NOs: 5-7), CAN20G2 (SEQ ID NOs: 21-23), CAN20G5 (SEQ ID NOs:37-39), or CAN20G8 (SEQ ID NOs: 53-55).

In certain embodiments, CDRs corresponding to the CDRs in Table 1 havesequence variations. For example, CDRs, in which 1, 2 3, 4, 5, 6, 7 or 8residues, or less than 20%, less than 30%, or less than about 40% oftotal residues in the CDR, are substituted or deleted can be present inan antibody (or antigen-binding portion thereof) that binds toxin A.

In one embodiment, the antibody or antigen-binding portion thereofcontains a variable light chain region and variable heavy chain regionhomologous to a variable light chain region and variable heavy chainregion of the antibody produced by clone CAN20G1 (SEQ ID NO: 4 and SEQID NO:12, respectively), CAN20G2 (SEQ ID NO:20 and SEQ ID NO:28,respectively), CAN20G5 (SEQ ID NO:36 and SEQ ID NO:44, respectively), orCAN20G8 (SEQ ID NO:52 and SEQ ID NO:60, respectively).

The antibodies or antigen-binding portions thereof are peptides. Thepeptides may also include variants, analogs, orthologs, homologs andderivatives of peptides, that exhibit a biological activity, e.g.,binding of an antigen. The peptides may contain one or more analogs ofan amino acid (including, for example, non-naturally occurring aminoacids, amino acids which only occur naturally in an unrelated biologicalsystem, modified amino acids from mammalian systems etc.), peptides withsubstituted linkages, as well as other modifications known in the art.

Also within the scope of the invention are antibodies or antigen-bindingportions thereof in which specific amino acids have been substituted,deleted or added. These alternations do not have a substantial effect onthe peptide's biological properties such as binding activity. Forexample, antibodies may have amino acid substitutions in the frameworkregion, such as to improve binding to the antigen. In another example, aselected, small number of acceptor framework residues can be replaced bythe corresponding donor amino acids. The donor framework can be a matureor germline human antibody framework sequence or a consensus sequence.Guidance concerning how to make phenotypically silent amino acidsubstitutions is provided in Bowie et al., Science, 247: 1306-1310(1990). Cunningham et al., Science, 244: 1081-1085 (1989). Ausubel(ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc.(1994). T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor,N.Y. (1989). Pearson, Methods Mol. Biol. 243:307-31 (1994). Gonnet etal., Science 256:1443-45 (1992).

The antibody, or antigen-binding portion thereof, can be derivatized orlinked to another functional molecule. For example, an antibody can befunctionally linked (by chemical coupling, genetic fusion, noncovalentinteraction, etc.) to one or more other molecular entities, such asanother antibody, a detectable agent, a cytotoxic agent, apharmaceutical agent, a protein or peptide that can mediate associationwith another molecule (such as a streptavidin core region or apolyhistidine tag), amino acid linkers, signal sequences, immunogeniccarriers, or ligands useful in protein purification, such asglutathione-S-transferase, histidine tag, and staphylococcal protein A.One type of derivatized protein is produced by crosslinking two or moreproteins (of the same type or of different types). Suitable crosslinkersinclude those that are heterobifunctional, having two distinct reactivegroups separated by an appropriate spacer (e.g.,m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional(e.g., disuccinimidyl suberate). Such linkers are available from PierceChemical Company, Rockford, Ill. Useful detectable agents with which aprotein can be derivatized (or labeled) include fluorescent compounds,various enzymes, prosthetic groups, luminescent materials,bioluminescent materials, and radioactive materials. Non-limiting,exemplary fluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, and, phycoerythrin. A protein or antibody canalso be derivatized with detectable enzymes, such as alkalinephosphatase, horseradish peroxidase, beta-galactosidase,acetylcholinesterase, glucose oxidase and the like. A protein can alsobe derivatized with a prosthetic group (e.g., streptavidin/biotin andavidin/biotin).

The present peptides may be the functionally active variant ofantibodies of antigen-binding portions thereof disclosed herein, e.g.,with less than about 30%, about 25%, about 20%, about 15%, about 10%,about 5% or about 1% amino acid residues substituted or deleted butretain essentially the same immunological properties including, but notlimited to, binding to toxin A.

The invention also encompasses a nucleic acid encoding the presentantibody or antigen-binding portion thereof that specifically binds totoxin A of C. difficile. The nucleic acid may be expressed in a cell toproduce the present antibody or antigen-binding portion thereof. Theisolated nucleic acid of the present invention comprises a sequenceencoding a peptide at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 99%, about 70%, about 75%, about 80%, about 81%, about82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 99% or about 100% homologousto SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52 or 60.

The isolated nucleic acid may comprise a sequence at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, at least about 99%, about 70%, about 75%, about80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%,about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100% homologous to SEQ ID NOs: 68, 69, 70, 71, 72, 73, 74 or 75.

The invention also features expression vectors including a nucleic acidencoding a peptide at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 99%, about 70%, about 75%, about 80%, about 81%, about82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 99% or about 100% homologousto SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52 or 60. The expression vectormay include a nucleic acid sequence at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 99%, about 70%, about 75%, about 80%,about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99% orabout 100% homologous to SEQ ID NOs: 68, 69, 70, 71, 72, 73, 74 or 75.

Nucleic acid molecules encoding a functionally active variant of thepresent antibody or antigen-binding portion thereof are also encompassedby the present invention. These nucleic acid molecules may hybridizewith a nucleic acid encoding any of the present antibody orantigen-binding portion thereof under medium stringency, highstringency, or very high stringency conditions. Guidance for performinghybridization reactions can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. 6.3.1-6.3.6, 1989, which isincorporated herein by reference. Specific hybridization conditionsreferred to herein are as follows: 1) medium stringency hybridizationconditions: 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 60° C.; 2) high stringency hybridizationconditions: 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 65° C.; and 3) very high stringency hybridizationconditions: 0.5 M sodium phosphate, 7% SDS at 65° C., followed by one ormore washes at 0.2×SSC, 1% SDS at 65° C.

A nucleic acid encoding the present antibody or antigen-binding portionthereof may be introduced into an expression vector that can beexpressed in a suitable expression system, followed by isolation orpurification of the expressed antibody or antigen-binding portionthereof. Optionally, a nucleic acid encoding the present antibody orantigen-binding portion thereof can be translated in a cell-freetranslation system. U.S. Pat. No. 4,816,567. Queen et al., Proc NatlAcad Sci USA, 86:10029-10033 (1989).

Anti-toxin antibodies or portions thereof can be produced by host cellstransformed with DNA encoding light and heavy chains (or portionsthereof) of a desired antibody. Antibodies can be isolated and purifiedfrom these culture supernatants and/or cells using standard techniques.For example, a host cell may be transformed with DNA encoding the lightchain, the heavy chain, or both, of an antibody. Recombinant DNAtechnology may also be used to remove some or all of the DNA encodingeither or both of the light and heavy chains that is not necessary forbinding, e.g., the constant region.

The present nucleic acids can be expressed in various suitable cells,including prokaryotic and eukaryotic cells, e.g., bacterial cells,(e.g., E. coli), yeast cells, plant cells, insect cells, and mammaliancells. A number of mammalian cell lines are known in the art and includeimmortalized cell lines available from the American Type CultureCollection (ATCC). Non-limiting examples of the cells include all celllines of mammalian origin or mammalian-like characteristics, includingbut not limited to, parental cells, derivatives and/or engineeredvariants of monkey kidney cells (COS, e.g., COS-1, COS-7), HEK293, babyhamster kidney (BHK, e.g., BHK21), Chinese hamster ovary (CHO), NS0,PerC6, BSC-1, human hepatocellular carcinoma cells (e.g., Hep G2),SP2/0, HeLa, Madin-Darby bovine kidney (MDBK), myeloma and lymphomacells. The engineered variants include, e.g., glycan profile modifiedand/or site-specific integration site derivatives.

The present invention also provides for cells comprising the nucleicacids described herein. The cells may be a hybridoma or transfectant.The types of the cells are discussed above.

The present antibody or antigen-binding portion thereof can be expressedin various cells. The types of the cells are discussed above.

Alternatively, the present antibody or antigen-binding portion thereofcan be synthesized by solid phase procedures well known in the art.Solid Phase Peptide Synthesis: A Practical Approach by E. Atherton andR. C. Sheppard, published by IRL at Oxford University Press (1989).Methods in Molecular Biology, Vol. 35: Peptide Synthesis Protocols (ed.M. W. Pennington and B. M. Dunn), chapter 7. Solid Phase PeptideSynthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill. (1984). G.Barany and R. B. Merrifield, The Peptides: Analysis, Synthesis, Biology,editors E. Gross and J. Meienhofer, Vol. 1 and Vol. 2, Academic Press,New York, (1980), pp. 3-254. M. Bodansky, Principles of PeptideSynthesis, Springer-Verlag, Berlin (1984).

The present invention provides for methods for making an antibody orantigen-binding portion thereof that specifically binds to toxin A of C.difficile. For example, a non-human animal is immunized with acomposition that includes an inactivated toxin A, and then a specificantibody is isolated from the animal. The method can further includeevaluating binding of the antibody to toxin A.

Any of a variety of Clostridium difficile toxin proteins, particularlytoxin A, may be used in the practice of the present invention. C.difficile disease is mediated primarily by toxin A and toxin B. Bothtoxins are cytotoxic, and lethal when injected intravenously orintraperitoneally into a mouse. Toxin A is also a potent enterotoxin, asdemonstrated by the induction of fluid accumulation in the mouse ligatedintestinal loop diarrhea model. See, e.g., Babcock, G. J. et al.,Infection and Immunity, 74: 6339-6347 (2006) and references containedtherein for background.

Table 2 provides amino acid sequences of Clostridium difficile toxin A.Variants and fragments of the sequences provided below can also be usedas an antigen to generate antibodies.

TABLE 2 SEQ ID Protein NO Name Amino acid Sequence 1 Toxin AMSLISKEELIKLAYSIRPRENEYKTILTNLDEYNKLTTNNNENKYLQLKKLNESIDVFMNKYKNSSRNRALSNLKKDILKEVILIKNSNTSPVEKNLHFVWIGGEVSDIALEYIKQWADINAEYNIKLWYDSEAFLVNTLKKAIVESSTTEALQLLEEEIQNPQFDNMKFYKKRMEFIYDRQKRFINYYKSQINKPTVPTIDDIIKSHLVSEYNRDETLLESYRTNSLRKINSNHGIDIRANSLFTEQELLNIYSQELLNRGNLAAASDIVRLLALKNFGGVYLDVDMLPGIHSDLFKTIPRPSSIGLDRWEMIKLEAIMKYKKYINNYTSENFDKLDQQLKDNFKLIIESKSEKSEIFSKLENLNVSDLEIKIAFALGSVINQALISKQGSYLTNLVIEQVKNRYQFLNQHLNPAIESDNNFTDTTKIFHDSLFNSATAENSMFLTKIAPYLQVGFMPEARSTISLSGPGAYASAYYDFINLQENTIEKTLKASDLIEFKFPENNLSQLTEQEINSLWSFDQASAKYQFEKYVRDYTGGSLSEDNGVDFNKNTALDKNYLLNNKIPSNNVEEAGSKNYVHYIIQLQGDDISYEATCNLFSKNPKNSIIIQRNMNESAKSYFLSDDGESILELNKYRIPERLKNKEKVKVTFIGHGKDEFNTSEFARLSVDSLSNEISSFLDTIKLDISPKNVEVNLLGCNMFSYDFNVEETYPGKLLLSIMDKITSTLPDVNKDSITIGANQYEVRINSEGRKELLAHSGKWINKEEAIMSDLSSKEYIFFDSIDNKLKAKSKNIPGLASISEDIKTLLLDASVSPDTKFILNNLKLNIESSIGDYIYYEKLEPVKNIIHNSIDDLIDEFNLLENVSDELYELKKLNNLDEKYLISFEDISKNNSTYSVRFINKSNGESVYVETEKEIFSKYSEHITKEISTIKNSIITDVNGNLLDNIQLDHTSQVNTLNAAFFIQSLIDYSSNKDVLNDLSTSVKVQLYAQLFSTGLNTIYDSIQLVNLISNAVNDTINVLPTITEGIPIVSTILDGINLGAAIKELLDEHDPLLKKELEAKVGVLAINMSLSIAATVASIVGIGAEVTIFLLPIAGISAGIPSLVNNELILHDKATSVVNYFNHLSESKEYGPLKTEDDKILVPIDDLVISEIDFNNNSIKLGTCNILAMEGGSGHTVTGNIDHFFSSPYISSHIPSLSVYSAIGIKTENLDFSKKIMMLPNAPSRVFWWETGAVPGLRSLENNGTKLLDSIRDLYPGKFYWRFYAFFDYAITTLKPVYEDTNTKIKLDKDTRNFIMPTITTDEIRNKLSYSFDGAGGTYSLLLSSYPISMNINLSKDDLWIFNIDNEVREISIENGTIKKGNLIEDVLSKIDINKNKLIIGNQTIDFSGDIDNKDRYIFLTCELDDKISLIIEINLVAKSYSLLLSGDKNYLISNLSNTIEKINTLGLDSKNIAYNYTDESNNKYFGAISKTSQKSIIHYKKDSKNILEFYNGSTLEFNSKDFIAEDINVFMKDDINTITGKYYVDNNTDKSIDFSISLVSKNQVKVNGLYLNESVYSSYLDFVKNSDGHHNTSNFMNLFLNNISFWKLFGFENINFVIDKYFTLVGKTNLGYVEFICDNNKNIDIYFGEWKTSSSKSTIFSGNGRNVVVEPIYNPDTGEDISTSLDFSYEPLYGIDRYINKVLIAPDLYTSLININTNYYSNEYYPEIIVLNPNTFHKKVNINLDSSSFEYKWSTEGSDFILVRYLEESNKKILQKIRIKGILSNTQSFNKMSIDFKDIKKLSLGYIMSNFKSFNSENELDRDHLGFKIIDNKTYYYDEDSKLVKGLININNSLFYFDPIESNLVTGWQTINGKKYYFDINTGAASTSYKIINGKHFYFNNNGVMQLGVFKGPDGFEYFAPANTQNNNIEGQAIVYQSKFLTLNGKKYYFDNDSKAVTGWRIINNEKYYFNPNNAIAAVGLQVIDNNKYYFNPDTAIISKGWQTVNGSRYYFDTDTAIAFNGYKTIDGKHFYFDSDCVVKIGVFSGSNGFEYFAPANTYNNNIEGQAIVYQSKFLTLNGKKYYFDNNSKAVTGWQTIDSKKYYFNTNTAEAATGWQTIDGKKYYFNTNTAEAATGWQTIDGKKYYFNTNTSIASTGYTIINGKYFYFNTDGIMQIGVFKVPNGFEYFAPANTHNNNIEGQAILYQNKFLTLNGKKYYFGSDSKAITGWQTIDGKKYYFNPNNAIAATHLCTINNDKYYFSYDGILQNGYITIERNNFYFDANNESKMVTGVFKGPNGFEYFAPANTHNNNIEGQAIVYQNKFLTLNGKKYYFDNDSKAVTGWQTIDSKKYYFNLNTAVAVTGWQTIDGEKYYFNLNTAEAATGWQTIDGKRYYFNTNTYIASTGYTIINGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTHNNNIEGQAILYQNKFLTLNGKKYYFGSDSKAVTGLRTIDGKKYYFNTNTAVAVTGWQTINGKKYYFNTNTYIASTGYTIISGKHFYFNTDGIMQIGVFKGPDGFEYFAPANTDANNIEGQAIRYQNRFLYLHDNIYYFGNDSKAATGWATIDGNRYYFEPNTAMGANGYKTIDNKNFYFRNGLPQIGVFKGPNGFEYFAPANTDANNIDGQAIRYQNRFLHLLGKIYYFGNNSKAVTGWQTINSKVYYFMPDTAMAA AGGLFEIDGVIYFFGVDGVKAPGIYG

Table 3 provides nucleic acid sequences encoding the proteins of Table2.

TABLE 3 Accession SEQ Number ID And Gene NO Name Nucleotide Sequence 2Toxin Aatgtctttaa tatctaaaga agagttaata aaactcgcat atagcattag accaagagaaaatgagtata aaactatact aactaattta gacgaatata ataagttaac tacaaacaataatgaaaata aatatttaca attaaaaaaa ctaaatgaat caattgatgt ttttatgaataaatataaaa attcaagcag aaatagagca ctctctaatc taaaaaaaga tatattaaaagaagtaattc ttattaaaaa ttccaataca agtcctgtag aaaaaaattt acattttgtatggataggtg gagaagtcag tgatattgct cttgaataca taaaacaatg ggctgatattaatgcagaat ataatattaa actgtggtat gatagtgaag cattcttagt caatacactaaaaaaggcta tagttgaatc ttctaccact gaagcattac agctactaga ggaagagattcaaaatcctc aatttgataa tatgaaattt tacaaaaaaa ggatggaatt tatatatgatagacaaaaaa ggtttataaa ttattataaa tctcaaatca ataaacctac agtacctacaatagatgata ttataaagtc tcatctagta tctgaatata atagagatga aactttattagaatcatata gaacaaattc tttgagaaaa ataaatagta atcatgggat agatatcagggctaatagtt tgtttacaga acaagagtta ttaaatattt atagtcagga gttgttaaatcgtgggaatt tagctgcagc atctgacata gtaagattat tagccctaaa aaattttggcggagtatatt tagatgttga tatgcttcca ggtattcact ctgatttatt taaaacaatacctagaccta gctctattgg actagaccgt tgggaaatga taaaattaga ggctattatgaagtataaaa aatatataaa taattataca tcagaaaact ttgataaact tgatcaacaattaaaagata attttaaact cattatagaa agtaaaagtg aaaaatctga gatattttctaaattagaaa atttaaatgt atctgatctt gaaattaaaa tagctttcgc tttaggcagtgttataaatc aagccttgat atcaaaacaa ggttcatatc ttactaacct agtaatagaacaagtaaaaa atagatatca atttttaaac caacacctta acccagccat agagtctgacaataacttca cagatactac taagattttt catgattcac tatttaattc agctaccgcagaaaactcta tgtttttaac aaaaatagca ccatacttac aagtaggttt tatgccagaagctcgctcca caataagttt aagtggtcca ggagcttatg catcagctta ctatgatttcataaatttac aagaaaatac tatagaaaaa actttaaaag catcagattt aatagaatttaaattcccag aaaataatct atctcaattg acagaacaag aaataaatag tctatggagctttgatcaag caagtgcaaa atatcaattt gagaaatatg taagagatta tactggtggatctctttctg aagacaatgg ggtagacttt aataaaaata ctgccctcga caaaaactatttattaaata ataaaattcc atcaaacaat gtagaagaag ctggaagtaa aaattatgttcattatatca tacagttaca aggagatgat ataagttatg aagcaacatg caatttattttctaaaaatc ctaaaaatag tattattata caacgaaata tgaatgaaag tgcaaaaagttactttttaa gtgatgatgg agaatctatt ttagaattaa ataaatatag gatacctgaaagattaaaaa ataaggaaaa agtaaaagta acctttattg gacatggtaa agatgaattcaacacaagcg aatttgctag attaagtgta gattcacttt ccaatgagat aagttcatttttagatacca taaaattaga tatatcacct aaaaatgtag aagtaaactt gcttggatgtaatatgttta gttatgattt taatgttgaa gaaacttatc ctggtaagtt actattaagtattatggaca aaattacttc cactttacct gatgtaaata aagattctat tactataggagcaaatcaat atgaagtaag aattaatagt gagggaagaa aagaacttct agctcactcaggtaaatgga taaataaaga ggaagctatt atgagcgatt tatctagtaa agaatacattttttttgatt ccatagataa taagctaaaa gcaaagtcca agaatattcc aggtttagcgtcaatatcag aagatataaa aacattatta cttgatgcaa gtgttagtcc tgatacaaaatttattttaa ataatcttaa gcttaatatt gaatcttcta ttggtgatta catttattatgaaaaattag aacctgttaa aaatataatc cacaattcta tagatgattt aatagatgagttcaatctac ttgaaaatgt atctgatgaa ttatatgaat taaaaaaatt aaataatctagatgagaagt atttaatatc ttttgaagat atctcaaaaa ataattcaac ttattctgtaagatttatta acaaaagtaa tggtgaatca gtttatgtag agacagaaaa agaaattttttcaaaatata gcgaacatat tacaaaagaa ataagtacta taaagaatag tataattacagatgttaatg gtaatttatt ggataatata cagttagatc atacttctca agttaatacattaaacgcag cattctttat tcaatcatta atagattata gtagcaataa agatgtactgaatgatttaa gtacctcagt taaggttcaa ctttatgctc aactatttag tacaggtttaaatactatat atgactctat ccaattagta aatttaatat caaatgcagt aaatgatactataaatgtac tacctacaat aacagagggg atacctattg tatctactat attagacggaataaacttag gtgcagcaat taaggaatta ctagacgaac atgacccatt actaaaaaaagaactagaag ctaaggtggg tgttttagca ataaatatgt cattatctat agctgcaacggtagcttcaa ttgttggaat aggtgctgaa gttactattt tcttattacc tatagctggtatatctgcgg gaataccttc attagttaat aatgaattaa tattgcatga taaggcaacttcagtggtaa actattttaa tcatttgtct gaatctaaag aatatggccc tcttaaaacagaagatgata aaattttagt tcctattgat gatttagtaa tatcagaaat agattttaataataattcga taaaactagg aacatgtaat atattagcaa tggagggggg atcaggacacacagtgactg gtaatataga tcactttttc tcatctccat atataagctc tcatattccttcattatcag tttattctgc aataggtata aaaacagaaa atctagattt ttcaaaaaaaataatgatgt taccaaatgc tccttcaaga gtgttttggt gggaaactgg agcagttccaggtttaagat cattggaaaa taatgggact aaattgcttg attcaataag agatttatacccaggcaaat tttactggag attctatgcc tttttcgatt atgcaataac tacattaaaaccagtgtatg aagacactaa tactaaaatt aaactagata aagatactag aaactttataatgccaacta taactactga cgaaattaga aacaaattat cttattcatt tgatggagcaggaggaactt actctttatt attatcttca tatccaatat caatgaatat aaatttatctaaagatgatt tatggatatt taatattgat aatgaagtaa gagaaatatc tatagaaaatggtactatta aaaaaggaaa tttaatagaa gatgttttaa gtaaaattga tataaataaaaataaactta ttataggcaa tcaaacaata gatttttcag gtgatataga taacaaagatagatatatat tcttgacttg tgagttagat gataaaatta gtttaataat agaaataaatcttgttgcaa aatcttatag tttgttattg tctggggata aaaattattt gatatccaatttatctaata ctattgagaa aatcaatact ttaggcctag atagtaaaaa tatagcttacaattacactg atgaatctaa taataaatat tttggagcta tatctaaaac aagtcaaaaaagcataatac attataaaaa agacagtaaa aatatattag aattttataa tggcagtacattagaattta acagtaaaga ctttattgct gaagatataa atgtatttat gaaagatgatattaatacta taacaggaaa atactatgtt gataataata ctgataaaag tatagatttctctatttctt tagttagtaa aaatcaagta aaagtaaatg gattatattt aaatgaatccgtatactcat cttaccttga ttttgtgaaa aattcagatg gacaccataa tacttctaattttatgaatt tatttttgaa caatataagt ttctggaaat tgtttgggtt tgaaaatataaattttgtaa tcgataaata ctttaccctt gttggtaaaa ctaatcttgg atatgtagaatttatttgtg acaataataa aaatatagat atatattttg gtgaatggaa aacatcgtcatctaaaagca ctatatttag cggaaatggt agaaatgttg tagtagagcc tatatataatcctgatacgg gtgaagatat atctacttca ctagattttt cctatgaacc tctctatggaatagatagat atatcaataa agtattgata gcacctgatt tatatacaag tttaataaatattaatacca attattattc aaatgagtac taccctgaga ttatagttct taacccaaatacattccaca aaaaagtaaa tataaattta gatagttctt cttttgagta taaatggtctacagaaggaa gtgactttat tttagttaga tacttagaag aaagtaataa aaaaatattacaaaaaataa gaatcaaagg tatcttatct aatactcaat catttaataa aatgagtatagattttaaag atattaaaaa actatcatta ggatatataa tgagtaattt taaatcatttaattctgaaa atgaattaga tagagatcat ttaggattta aaataataga taataaaacttattactatg atgaagatag taaattagtt aaaggattaa tcaatataaa taattcattattctattttg atcctataga atctaactta gtaactggat ggcaaactat caatggtaaaaaatattatt ttgatataaa tactggagca gcttcaacta gttataaaat tattaatggtaaacactttt attttaataa taatggtgtg atgcagttag gagtatttaa aggacctgatggatttgagt attttgcacc tgccaatact cagaataata acatagaagg tcaggctatagtttatcaaa gtaaattctt aactttgaat ggcaaaaaat attattttga taatgactcaaaagcagtca ctggatggag gattattaac aatgagaaat attactttaa tcctaataatgctattgctg cagtcggatt gcaagtaatt gacaataata agtattattt caatcctgacactgctatca tctcaaaagg ttggcagact gttaatggta gtagatacta ctttgatactgataccgcta ttgcctttaa tggttataaa actattgatg gtaaacactt ttattttgatagtgattgtg tagtgaaaat aggtgtgttt agtggctcta atggatttga atatttcgcacctgctaata cttataataa taacatagaa ggtcaggcta tagtttatca aagtaaattcttaactttga atggtaaaaa atattacttt gataataact caaaagcagt taccggatggcaaactattg atagtaaaaa atattacttt aatactaaca ctgctgaagc agctactggatggcaaacta ttgatggtaa aaagtattac tttaatacta acactgctga agcagctactggatggcaaa ctattgatgg taaaaaatat tactttaata ctaacacttc tatagcttcaactggttata caattattaa tggtaaatat ttttatttta atactgatgg tattatgcagataggagtgt ttaaagtacc taatggattt gaatactttg cacctgctaa tactcataataataacatag aaggtcaagc tatactttac caaaataaat tcttaacttt gaatggtaaaaaatattact ttggtagtga ctcaaaagca attactggat ggcaaaccat tgatggtaaaaaatattact ttaatcctaa taatgctatt gctgcgactc atctatgcac tataaataacgacaagtatt actttagtta tgatggaatt cttcaaaatg gatatattac tattgaaagaaataatttct attttgatgc taataatgaa tctaaaatgg taacaggagt atttaaaggacctaatggat ttgagtattt tgcacctgct aatactcata ataataacat agaaggtcaggctatagttt accagaataa attcttaact ttgaatggca aaaaatatta ttttgataatgactcaaaag cagttactgg atggcaaact attgatagta aaaaatatta ctttaatctt aacactgctg ttgcagttac tggatggcaa actattgatg gtgaaaaata ttactttaat cttaacactg ctgaagcagc tactggatgg caaactattg atggtaaaag atactacttt aatactaaca cttatatagc ttcaactggt tatacgatta ttaatggtaa acatttttat tttaatactg atggtattat gcagatagga gtgtttaaag gacctgatgg atttgaatac tttgcacctg ctaatactca taataataac atagaaggtc aagctatact ttaccaaaat aaattcttaa ctttgaatgg taaaaaatat tactttggta gtgactcaaa agcagttacc ggattgcgaa ctattgatgg taaaaaatat tactttaata ctaacactgc tgttgcagtt actggatggc aaactattaa tggtaaaaaa tactacttta atactaacac ttatatagct tcaactggtt atacaattat tagtggtaaa catttttatt ttaatactga tggtattatg cagataggag tgtttaaagg acctgatgga tttgaatact ttgcacctgc taatacggat gctaacaaca tagaaggtca agctatacgt tatcaaaata gattcctata tttacatgac aatatatatt actttggcaa tgattcaaaa gcggctactg gttgggcaac tattgatggt aatagatatt acttcgagcc taatacagct atgggtgcga atggttataa aactattgat aataaaaatt tttactttag aaatggttta cctcagatag gagtgtttaa aggacctaat ggatttgaat actttgcacc tgctaatacg gatgctaaca atatagatgg tcaagctata cgttatcaaa atagattcct acatttactt ggaaaaatat attactttgg taataactca aaagcagtta ctggatggca aactattaat agtaaagtat attactttat gcctgatact gctatggctg cagctggtgg acttttcgag attgatggtg ttatatattt ctttggtgtt gatggagtaa aagcccctgg gatatatggc taa 

In one embodiment, the present invention provides for a method formaking a hybridoma that expresses an antibody that specifically binds totoxin A of C. difficile. The method contains the following steps:immunizing an animal with a composition that includes inactivated toxinA (e.g., toxoid A); isolating splenocytes from the animal; generatinghybridomas from the splenocytes; and selecting a hybridoma that producesan antibody that specifically binds to toxin A. Kohler and Milstein,Nature, 256: 495, 1975. Harlow, E. and Lane, D. Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1988.

Toxins can be inactivated, for example, by treatment with formaldehyde,glutaraldehyde, UDP-dialdehyde, peroxide, oxygen or by mutation (e.g.,using recombinant methods). Relyveld et al., Methods in Enzymology,93:24, 1983. Woodrow and Levine, eds., New Generation Vaccines, MarcelDekker, Inc., New York, 1990. Genth et al., Inf. and Immun.,68(3):1094-1101, 2000. Mutant C. difficile toxins with reduced toxicitycan be produced using recombinant methods. U.S. Pat. Nos. 5,085,862;5,221,618; 5,244,657; 5,332,583; 5,358,868; and 5,433,945. A full-lengthor fragment of the toxins or toxoids can be used as immunogens.

In one embodiment, inactivated toxin A is used to immunize miceintraperitoneally or intravenously. One or more boosts may or may not begiven. The titers of the antibodies in the plasma can be monitored by,e.g., ELISA (enzyme-linked immunosorbent assay) or flow cytometry. Micewith sufficient titers of anti-toxin A antibodies are used for fusions.Mice may or may not be boosted with antigen 3 days before sacrifice andremoval of the spleen. The mouse splenocytes are isolated and fused withPEG to a mouse myeloma cell line. The resulting hybridomas are thenscreened for the production of antigen-specific antibodies. Cells areplated, and then incubated in selective medium. Supernatants fromindividual wells are then screened by ELISA for human anti-toxinmonoclonal antibodies. The antibody secreting hybridomas are replated,screened again, and if still positive for anti-toxin monoclonalantibodies, can be subcloned by limiting dilution. For example, thehybridoma clone CAN20G2 of the present invention has been subcloned. Oneof the subclones is CAN20G2-2-1.

Adjuvants that may be used to increase the immunogenicity of one or moreof the Clostridium difficile toxin antigens, particularly toxin Ainclude any compound or compounds that act to increase an immuneresponse to peptides or combination of peptides. Non-limiting examplesof adjuvants include alum, aluminum phosphate, aluminum hydroxide, MF59(4.3% w/v squalene, 0.5% w/v polysorbate 80 (Tween 80), 0.5% w/vsorbitan trioleate (Span 85)), CpG-containing nucleic acid, QS21(saponin adjuvant), MPL (Monophosphoryl Lipid A), 3DMPL (3-O-deacylatedMPL), extracts from Aquilla, ISCOMS (see, e.g., Sjolander et al. (1998)J. Leukocyte Biol. 64:713; WO90/03184; WO96/11711; WO 00/48630;WO98/36772; WO00/41720; WO06/134423 and WO07/026190), LT/CT mutants,poly(D,L-lactide-co-glycolide) (PLG) microparticles, Quil A,interleukins, Freund's, N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637,referred to as nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dip-almitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion.

The immunized animal can be any animal that is capable of producingrecoverable antibodies when administered an immunogen, such as, but notlimited to, rabbits, mice, rats, hamsters, goats, horses, monkeys,baboons and humans. In one aspect, the host is transgenic and produceshuman antibodies, e.g., a mouse expressing the human immunoglobulin genesegments. U.S. Pat. Nos. 8,236,311; 7,625,559 and 5,770,429, thedisclosure of each of which is incorporated herein by reference in itsentirety. Lonberg et al., Nature 368(6474): 856-859, 1994. Lonberg, N.,Handbook of Experimental Pharmacology 113:49-101, 1994. Lonberg, N. andHuszar, D., Intern. Rev. Immunol., 13: 65-93, 1995. Harding, F. andLonberg, N., Ann. N.Y. Acad. Sci., 764:536-546, 1995.

After the host is immunized and the antibodies are produced, theantibodies are assayed to confirm that they are specific for the antigenof interest and to determine whether they exhibit any cross reactivitywith other antigens. One method of conducting such assays is a serascreen assay as described in U.S. Patent Publication No. 2004/0126829.Anti-toxin antibodies can be characterized for binding to the toxin by avariety of known techniques. For example, in an ELISA, microtiter platesare coated with the toxin or toxoid antigen in PBS, and then blockedwith irrelevant proteins such as bovine serum albumin (BSA) diluted inPBS. Dilutions of plasma from toxin-immunized mice are added to eachwell and incubated. The plates are washed and then incubated with asecondary antibody conjugated to an enzyme (e.g., alkaline phosphatase).After washing, the plates are developed with the enzyme's substrate(e.g., ABTS), and analyzed at a specific OD. In other embodiments, todetermine if the selected monoclonal antibodies bind to unique epitopes,the antibody can be biotinylated which can then be detected with astreptavidin labeled probe. Anti-toxin antibodies can be tested forreactivity with the toxin by Western blotting.

Neutralization assays can also be used to measure activity of theanti-toxin antibodies. For example, in vitro neutralization assays canbe used to measure the ability of an antibody to inhibit a cytopathiceffect on cells in culture (see Examples 7 and 12 below). In oneembodiment, the present antibody, or antigen-binding portion thereof, ata concentration ranging from about 1 μM to about 50 μM, from about 2 μMto about 40 μM, from about 3 μM to about 30 μM, from about 4 μM to about20 μM, from about 4 μM to about 17 μM, from about 5 μM to about 15 μM,or about 10 μM neutralizes greater than about 20%, greater than about30%, greater than about 40%, greater than about 50%, greater than about60%, greater than about 70%, greater than about 80%, or greater thanabout 90% of about 150 ng/ml C. difficile toxin A in an in vitroneutralization assay. In vivo assays can be used to measure toxinneutralization as well. In another embodiment, in an in vivo toxin Achallenge experiment (e.g., procedures as described in Examples 5, 6,and 7, as well as Babcock et al., Human Monoclonal Antibodies Directedagainst Toxins A and B prevent Clostridium difficile-Induced Mortalityin Hamsters. Infection and Immunity (2006) 74(11):6339), when theantibody, or an antigen-binding portion thereof, is administered to amammal at a dosage ranging from about 1 mg/kg body weight to about 50mg/kg body weight, from about 2 mg/kg body weight to about 40 mg/kg bodyweight, from about 3 mg/kg body weight to about 30 mg/kg body weight,from about 5 mg/kg body weight to about 20 mg/kg body weight, from about8 mg/kg body weight to about 13 mg/kg body weight, or about 10 mg/kgbody weight about 24 hours before the mammal is exposed to greater thanabout 100 ng, or about 100 ng of C. difficile toxin A, the chance ofsurvival for the mammal is greater than about 40%, greater than about50%, greater than about 60%, greater than about 70%, greater than about80%, or greater than about 90% within about 7 days.

Hybridomas that produce antibodies that bind, preferably with highaffinity, to the toxin can than be subcloned and further characterized.One clone from each hybridoma, which retains the reactivity of theparent cells (by ELISA), can then be chosen for making a cell bank, andfor antibody purification.

To purify the anti-toxin antibodies, supernatants from the culturedhybridomas can be filtered and concentrated before affinitychromatography with protein A-Sepharose (Pharmacia, Piscataway, N.J.).

Antibodies, or antigen-binding fragments, variants or derivativesthereof of the present disclosure can also be described or specified interms of their binding affinity to an antigen. The affinity of anantibody for an antigen can be determined experimentally using anysuitable method (see, e.g., Berzofsky et al., “Antibody-AntigenInteractions,” In Fundamental Immunology, Paul, W. E., Ed., Raven Press:New York, N.Y. (1984); Kuby, Janis Immunology, W. H. Freeman andCompany: New York, N.Y. (1992); and methods described herein). Themeasured affinity of a particular antibody-antigen interaction can varyif measured under different conditions (e.g., salt concentration, pH).Thus, measurements of affinity and other antigen-binding parameters(e.g., K_(D), K_(a), K_(d)) are preferably made with standardizedsolutions of antibody and antigen, and a standardized buffer.

The present antibodies or antigen-binding portions thereof have in vitroand in vivo therapeutic, prophylactic, and/or diagnostic utilities. Forexample, these antibodies can be administered to cells in culture, e.g.,in vitro or ex vivo, or to a subject, e.g., in vivo, to treat, inhibit,prevent relapse, and/or diagnose C. difficile and disease associatedwith C. difficile.

The antibodies or antigen-binding portions thereof can be used on cellsin culture, e.g., in vitro or ex vivo. For example, cells can becultured in vitro in culture medium and contacted by the anti-toxinantibody or fragment thereof. The methods can be performed on cellspresent in a subject, as part of an in vivo (e.g., therapeutic orprophylactic) protocol. For in vivo embodiments, the contacting step iseffected in a subject and includes administering an anti-toxin antibodyor portion thereof to the subject under conditions effective to permitbinding of the antibody, or portion thereof, to a toxin (e.g., toxin A)expressed by C. difficile in the subject, e.g., in the gut.

The antibody or antigen-binding portion thereof can be administeredalone or in combination with another therapeutic agent, e.g., a secondmonoclonal or polyclonal antibody or antigen-binding portion thereof. Inone example, the antibody or antigen-binding portion thereofspecifically binds to C. difficile toxin A is combined with a antibody(monoclonal or polyclonal) or antigen-binding portion thereofspecifically binds to C. difficile toxin B. In another example, thesecond agent is an antibiotic, e.g., vancomycin, bacitracin ormetronidazole. The antibodies can be used in combination with probioticagents such as Saccharomyces boulardii. The antibodies can also beadministered in combinations with a C. difficile vaccine, e.g., a toxoidvaccine.

The present invention also provides compositions containing an antibodyor antigen-binding portion thereof described herein, and apharmaceutically acceptable carrier. The composition may contain anisolated nucleic acid encoding the present antibody or antigen-bindingportion thereof, and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers include any and all solvents,dispersion media, isotonic and absorption delaying agents, and the likethat are physiologically compatible. In one embodiment, the compositionis effective to reduce, eliminate, or prevent Clostridium difficilebacterial infection in a subject.

The invention also features methods of treating C. difficile disease ina subject by administering to the subject the present antibody orantigen-binding portion thereof in an amount effective to inhibit C.difficile disease. Routes of administration of the present compositionsinclude, but are not limited to, intravenous, intramuscular,subcutaneous, oral, topical, subcutaneous, intradermal, transdermal,subdermal, parenteral, rectal, spinal, or epidermal administration.

The compositions of the present invention can be prepared asinjectables, either as liquid solutions or suspensions, or as solidforms which are suitable for solution or suspension in liquid vehiclesprior to injection. The composition can also be prepared in solid form,emulsified or the active ingredient encapsulated in liposome vehicles orother particulate carriers used for sustained delivery. For example, thecomposition can be in the form of an oil emulsion, water-in-oilemulsion, water-in-oil-in-water emulsion, site-specific emulsion,long-residence emulsion, stickyemulsion, microemulsion, nanoemulsion,liposome, microparticle, microsphere, nanosphere, nanoparticle andvarious natural or synthetic polymers, such as nonresorbable impermeablepolymers such as ethylenevinyl acetate copolymers and Hytrel®copolymers, swellable polymers such as hydrogels, or resorbable polymerssuch as collagen and certain polyacids or polyesters such as those usedto make resorbable sutures, that allow for sustained release of thevaccine.

The present antibodies or antigen-binding portions thereof areformulated into compositions for delivery to a mammalian subject. Thecomposition is administered alone, and/or mixed with a pharmaceuticallyacceptable vehicle or excipient. Suitable vehicles are, for example,water, saline, dextrose, glycerol, ethanol, or the like, andcombinations thereof. In addition, the vehicle can contain minor amountsof auxiliary substances such as wetting or emulsifying agents, pHbuffering agents, or adjuvants. The compositions of the presentinvention can also include ancillary substances, such as pharmacologicalagents, cytokines, or other biological response modifiers.

Furthermore, the compositions can be formulated into compositions ineither neutral or salt forms. Pharmaceutically acceptable salts includethe acid addition salts (formed with the free amino groups of the activepolypeptides) and which are formed with inorganic acids such as, forexample, hydrochloric or phosphoric acids, or organic acids such asacetic, oxalic, tartaric, mandelic, and the like. Salts formed from freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 21stedition.

Compositions can be administered in a single dose treatment or inmultiple dose treatments on a schedule and over a time periodappropriate to the age, weight and condition of the subject, theparticular composition used, and the route of administration.

In one embodiment, a single dose of the composition according to theinvention is administered. In other embodiments, multiple doses areadministered. The frequency of administration can vary depending on anyof a variety of factors, e.g., severity of the symptoms, degree ofimmunoprotection desired, whether the composition is used forprophylactic or curative purposes, etc. For example, in one embodiment,the composition according to the invention is administered once permonth, twice per month, three times per month, every other week (qow),once per week (qw), twice per week (biw), three times per week (tiw),four times per week, five times per week, six times per week, everyother day (qod), daily (qd), twice a day (qid), or three times a day(tid).

The duration of administration of a polypeptide according to theinvention, e.g., the period of time over which the composition isadministered, can vary, depending on any of a variety of factors, e.g.,subject response, etc. For example, the composition can be administeredover a period of time ranging from about one day to about one week, fromabout two weeks to about four weeks, from about one month to about twomonths, from about two months to about four months, from about fourmonths to about six months, from about six months to about eight months,from about eight months to about 1 year, from about 1 year to about 2years, or from about 2 years to about 4 years, or more.

The compositions can be combined with a pharmaceutically acceptablecarrier (excipient) to form a pharmacological composition.Pharmaceutically acceptable carriers can contain a physiologicallyacceptable compound that acts to, e.g., stabilize, or increase ordecrease the absorption or clearance rates of the pharmaceuticalcompositions of the invention. Physiologically acceptable compounds caninclude, e.g., carbohydrates, such as glucose, sucrose, or dextrans,antioxidants, such as ascorbic acid or glutathione, chelating agents,low molecular weight proteins, detergents, liposomal carriers, orexcipients or other stabilizers and/or buffers. Other physiologicallyacceptable compounds include wetting agents, emulsifying agents,dispersing agents or preservatives. See e.g., the 21st edition ofRemington's Pharmaceutical Science, Mack Publishing Company, Easton, Pa.(“Remington's”).

In one aspect, a solution of the composition are dissolved in apharmaceutically acceptable carrier, e.g., an aqueous carrier if thecomposition is water-soluble. Examples of aqueous solutions include,e.g., water, saline, phosphate buffered saline, Hank's solution,Ringer's solution, dextrose/saline, glucose solutions and the like. Theformulations can contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such asbuffering agents, tonicity adjusting agents, wetting agents, detergentsand the like. Additives can also include additional active ingredientssuch as bactericidal agents, or stabilizers. For example, the solutioncan contain sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate or triethanolamineoleate.

Solid formulations can be used in the present invention. They can beformulated as, e.g., pills, tablets, powders or capsules. For solidcompositions, conventional solid carriers can be used which include,e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharin,talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.Suitable pharmaceutical excipients include e.g., starch, cellulose,talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, magnesium stearate, sodium stearate, glycerol monostearate,sodium chloride, dried skim milk, glycerol, propylene glycol, water,ethanol.

When administered orally, the present compositions may be protected fromdigestion. This can be accomplished either by complexing the antibody orantigen-binding portion thereof with a composition to render itresistant to acidic and enzymatic hydrolysis or by packaging theantibody or antigen-binding portion thereof in an appropriatelyresistant carrier such as a liposome. Means of protecting compounds fromdigestion are well known in the art. Fix, Pharm Res. 13: 1760-1764,1996. Samanen, J. Pharm. Pharmacol. 48: 119-135, 1996. U.S. Pat. No.5,391,377.

For transmucosal or transdermal administration, penetrants appropriateto the barrier to be permeated can be used in the formulation. Suchpenetrants are generally known in the art, and include, e.g., fortransmucosal administration, bile salts and fusidic acid derivatives. Inaddition, detergents can be used to facilitate permeation. Transmucosaladministration can be through nasal sprays or using suppositories.Sayani, Crit. Rev. Ther. Drug Carrier Syst. 13: 85-184, 1996. Fortopical, transdermal administration, the agents are formulated intoointments, creams, salves, powders and gels. Transdermal deliverysystems can also include, e.g., patches.

The present compositions can also be administered in sustained deliveryor sustained release mechanisms. For example, biodegradeablemicrospheres or capsules or other biodegradeable polymer configurationscapable of sustained delivery of a peptide can be included in theformulations of the invention (see, e.g., Putney, Nat. Biotechnol. 16:153-157, 1998).

For inhalation, the present compositions can be delivered using anysystem known in the art, including dry powder aerosols, liquids deliverysystems, air jet nebulizers, propellant systems, and the like. Patton,Biotechniques 16: 141-143, 1998. Also can be used in the presentinvention are product and inhalation delivery systems for polypeptidemacromolecules by, e.g., Dura Pharmaceuticals (San Diego, Calif.),Aradigrn (Hayward, Calif.), Aerogen (Santa Clara, Calif.), InhaleTherapeutic Systems (San Carlos, Calif.), and the like. For example, thepharmaceutical formulation can be administered in the form of an aerosolor mist. For aerosol administration, the formulation can be supplied infinely divided form along with a surfactant and propellant. In anotheraspect, the device for delivering the formulation to respiratory tissueis an inhaler in which the formulation vaporizes. Other liquid deliverysystems include, e.g., air jet nebulizers.

Compositions or nucleic acids, polypeptides, or antibodies of theinvention can be delivered alone or as pharmaceutical compositions byany means known in the art, e.g., systemically, regionally, or locally;by intra-arterial, intrathecal (IT), intravenous (IV), parenteral,intra-pleural cavity, topical, oral, or local administration, assubcutaneous, intra-tracheal (e.g., by aerosol) or transmucosal (e.g.,buccal, bladder, vaginal, uterine, rectal, nasal mucosa). For a“regional effect,” e.g., to focus on a specific organ, one mode ofadministration includes intra-arterial or intrathecal (IT) injections,e.g., to focus on a specific organ, e.g., brain and CNS (see e.g.,Gurun, Anesth Analg. 85: 317-323, 1997). For example, intra-carotidartery injection can be used where it is desired to deliver a nucleicacid, peptide or polypeptide of the invention directly to the brain.Actual methods for preparing parenterally administrable compositionswill be known or apparent to those skilled in the art and are describedin detail. Bai, J. Neuroimmunol. 80: 65-75, 1997. Warren, J. Neurol.Sci. 152: 31-38, 1997. Tonegawa, J. Exp. Med. 186: 507-515, 1997.

In one aspect, the pharmaceutical formulations comprising compositionsor nucleic acids, polypeptides, or antibodies of the invention areincorporated in lipid monolayers or bilayers, e.g., liposomes. U.S. Pat.Nos. 6,110,490; 6,096,716; 5,283,185 and 5,279,833. Aspects of theinvention also provide formulations in which water soluble nucleicacids, peptides or polypeptides of the invention have been attached tothe surface of the monolayer or bilayer. For example, peptides can beattached to hydrazide-PEG-(distearoylphosphatidyl)ethanolamine-containing liposomes (see, e.g., Zalipsky, Bioconjug. Chem.6: 705-708, 1995). Liposomes or any form of lipid membrane, such asplanar lipid membranes or the cell membrane of an intact cell, e.g., ared blood cell, can be used. Liposomal formulations can be by any means,including administration intravenously, transdermally (see, e.g., Vutla,J. Pharm. Sci. 85: 5-8, 1996), transmucosally, or orally. The inventionalso provides pharmaceutical preparations in which the nucleic acid,peptides and/or polypeptides of the invention are incorporated withinmicelles and/or liposomes (see, e.g., Suntres, J. Pharm. Pharmacol. 46:23-28, 1994; Woodle, Pharm. Res. 9: 260-265, 1992). Liposomes andliposomal formulations can be prepared according to standard methods andare also well known in the art. Akimaru, Cytokines Mol. Ther. 1:197-210, 1995. Alving, Immunol. Rev. 145: 5-31, 1995. Szoka, Ann. Rev.Biophys. Bioeng. 9: 467, 1980. U.S. Pat. Nos. 4,235,871; 4,501,728 and4,837,028.

In one aspect, the compositions are prepared with carriers that willprotect the peptide against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Methods for preparationof such formulations will be apparent to those skilled in the art.Liposomal suspensions can also be used as pharmaceutically acceptablecarriers. U.S. Pat. No. 4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. In oneembodiment, the dosage of such compounds lies within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. Inanother embodiment, the therapeutically effective dose can be estimatedinitially from cell culture assays. A dose can be formulated in animalmodels to achieve a circulating plasma concentration range that includesthe IC₅₀ (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture.Sonderstrup, Springer, Sem. Immunopathol. 25: 35-45, 2003. Nikula etal., Inhal. Toxicol. 4(12): 123-53, 2000.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antigen-bindingportion of the invention is from about 0.001 to about 60 mg/kg bodyweight, about 0.01 to about 30 mg/kg body weight, about 0.01 to about 25mg/kg body weight, about 0.5 to about 25 mg/kg body weight, about 0.1 toabout 20 mg/kg body weight, about 10 to about 20 mg/kg body weight,about 0.75 to about 10 mg/kg body weight, about 1 to about 10 mg/kg bodyweight, about 2 to about 9 mg/kg body weight, about 1 to about 2 mg/kgbody weight, about 3 to about 8 mg/kg body weight, about 4 to about 7mg/kg body weight, about 5 to about 6 mg/kg body weight, about 8 toabout 13 mg/kg body weight, about 8.3 to about 12.5 mg/kg body weight,about 4 to about 6 mg/kg body weight, about 4.2 to about 6.3 mg/kg bodyweight, about 1.6 to about 2.5 mg/kg body weight, about 2 to about 3mg/kg body weight, or about 10 mg/kg body weight.

The composition is formulated to contain an effective amount of thepresent antibody or antigen-binding portion thereof, wherein the amountdepends on the animal to be treated and the condition to be treated. Inone embodiment, the present antibody or antigen-binding portion thereofis administered at a dose ranging from about 0.01 mg to about 10 g, fromabout 0.1 mg to about 9 g, from about 1 mg to about 8 g, from about 1 mgto about 7 g, from about 5 mg to about 6 g, from about 10 mg to about 5g, from about 20 mg to about 1 g, from about 50 mg to about 800 mg, fromabout 100 mg to about 500 mg, from about 0.01 mg to about 10 g, fromabout 0.05 μg to about 1.5 mg, from about 10 μg to about 1 mg protein,from about 30 μg to about 500 μg, from about 40 pg to about 300 pg, fromabout 0.1 μg to about 200 mg, from about 0.1 μg to about 5 from about 5μg to about 10 μg, from about 10 μg to about 25 μg, from about 25 μg toabout 50 μg, from about 50 μg to about 100 μg, from about 100 μg toabout 500 from about 500 μg to about 1 mg, from about 1 mg to about 2mg. The specific dose level for any particular subject depends upon avariety of factors including the activity of the specific peptide, theage, body weight, general health, sex, diet, time of administration,route of administration, and rate of excretion, drug combination and theseverity of the particular disease undergoing therapy.

In therapeutic applications, the present compositions are administeredto a subject at risk for Clostridium difficile bacterial infection orsuffering from active infection in an amount sufficient to at leastpartially arrest or prevent the condition or a disease and/or itscomplications.

An anti-toxin antibody (e.g., monoclonal antibody) can also be used toisolate toxins by standard techniques, such as affinity chromatographyor immunoprecipitation. Moreover, an anti-toxin antibody can be used todetect the toxin, e.g., to screen samples (e.g., in a stool sample) forthe presence of C. difficile. Anti-toxin antibodies can be useddiagnostically to monitor levels of the toxin in tissue as part of aclinical testing procedure, e.g., to, for example, determine theefficacy of a given treatment regimen.

The invention also provides kits containing an anti-toxin antibody orantigen-binding portion thereof. Additional components of the kits mayinclude one or more of the following: instructions for use; otherreagents, a therapeutic agent, or an agent useful for coupling anantibody to a label or therapeutic agent, or other materials forpreparing the antibody for administration; pharmaceutically acceptablecarriers; and devices or other materials for administration to asubject.

Various combinations of antibodies can be packaged together. Forexample, a kit can include antibodies that bind to toxin A andantibodies that bind to toxin B (e.g., monoclonal anti-toxin Bantibodies, or polyclonal antisera reactive with toxin B). Theantibodies can be mixed together, or packaged separately within the kit.

Instructions for use can include instructions for therapeuticapplication including suggested dosages and/or modes of administration,e.g., in a patient with a symptom of CDAD. Other instructions caninclude instructions on coupling of the antibody to a label or atherapeutic agent, or for purification of a conjugated antibody, e.g.,from unreacted conjugation components.

The kit may or may not contain at least one nucleic acid encodinganti-toxin antibodies or fragment thereof, and instructions forexpression of the nucleic acids. Other possible components of the kitinclude expression vectors and cells.

The present antibodies, antigen-binding portions thereof, compositionsand methods can be used in all vertebrates, e.g., mammals andnon-mammals, including human, mice, rats, guinea pigs, hamsters, dogs,cats, cows, horses, goats, sheep, pigs, monkeys, apes, gorillas,chimpanzees, rabbits, ducks, geese, chickens, amphibians, reptiles andother animals.

The following examples of specific aspects for carrying out the presentinvention are offered for illustrative purposes only, and are notintended to limit the scope of the present invention in any way.

EXAMPLES Example 1 Hybridoma Fusion

A classical hybridoma fusion was performed. Mice receive their firstimmunization with toxoid A using Complete Freund's Adjuvant (CFA) andtwo subsequent boosters on days 28 and 48 with toxoid A and IncompleteFreund's Adjuvant (IFA). A trial bleed was performed at day 55 and theserum was tested to check for titres of anti-toxoid A antibody. If IgGtitres were high enough fusions were performed. If not, mice receivedtwo more boosts with IFA and a second trial bleed was taken. Fusionswere performed using 2 mice at a time. Mice were given a final pushintraperitoneally (i.p.) with toxoid A in PBS three days prior to thefusion.

On the day of the fusion, mice are sacrificed and their spleens removed.Splenocytes are washed from the spleen using a syringe and needle andcollected in a 50 ml tube for fusion with myeloma cells. Myelomas are animmortal tumor cell line used as fusion partners, grown in the presenceof 8-azaguanine, a toxic nucleotide analog which blocks the salvagepathway. Cells grown in the presence of 8-aza survive only by incurringdefective mutations in the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) gene. B cells are fused with the myeloma cells usingPolyethylene Glycol (PEG) 1500. Fused cells are mixed into semi-solidagarose with drug selection and plated out into petri dishes. HAT mediacontaining Hypoxanthine, Aminopterin, and Thymidine is used for drugselection. Aminopterin is a drug which inhibits the de novo pathway fornucleotide metabolism which is absolutely required for survival/cellgrowth in myeloma lines defective in HGPRT, and allows selection usuallywithin 24-48 hours.

Example 2 Hybridoma Screening

The next step is screening of the growing hybridomas. A commercialsemisolid agarose within which the cells grow as “balls” of cells in the3-D matrix was used. This facilitates the picking of these balls by hand(by visual inspection) and transferring these clonal balls into a 96well plate containing suitable media. The cells were allowed to grow for3-7 days and then the supernatant removed for screening and replacedwith fresh media. Positive binding in ELISA (or other tests) resulted incontinuing to grow the hybridomas by transferring them up into largertissue culture vessels with increasing volume. The mAbs were isotypedusing a suitable commercial isotyping kit for murine mAbs using thespent supernatant. The decision to move a clone to the next stage ofselection is based on its reactivity to native toxin A using an ELISAand its survival, usually based upon serial dilutions and reactivity ofat least ⅛ or 1/16 or higher, as well as IgG class; therefore the numberof clones decreased throughout the selection procedure. The mAbs thatunderwent further characterization were: CAN20G2, CAN20G1, CAN20G5 &CAN20G8 and CAN19G1, CAN19G2, CAN19G3.

Example 3 ELISA Assay of Mouse Monoclonal Antibodies

An ELISA was used to test the binding of the toxA mAbs against wholetoxin A and recombinant toxin A fragment 4 as well as to determine ifthey were cross-reactive to whole toxin B and toxin B fragment 4. ThemAb clones were compared to CDA1 (Merck anti-toxin A mAb used as acontrol). The ELISA plate was coated with 100 μg/ml of Toxin fragment 4and 400 μg/ml of whole Toxin so that the coatings were equimolar. Thewells were blocked with 1% skim milk then probed with serially dilutedCAN19 or CAN20 mAbs (0.1 μg/ml to 1 μg/ml) and binding was detected witha commercial goat anti-mouse IgG-HRP antibody. Negative and positivecontrols were also run. The chimeric human mAb 13C6 is specific toEbolavirus GP and served as the negative control for human the Fc ofCDA-1. The CDA-1 mAb and the polyclonal toxoid A antibody (pAb) servedas positive controls, however, the CDA-1 mAb is human and the polyclonalis rabbit, thus, they both used a different secondary antibody makingdirect comparisons between them and the murine mAbs impossible. Thesecondary antibody control is for the murine secondary antibody. Theplate was read at 405 nm after 60 min incubation with substrate. Thetitration data for each antibody is shown in FIG. 1.

Results:

As shown in FIGS. 2 and 3, CAN19G1 and CAN19G2 mAbs bind to whole toxinA and toxin A fragment 4 at a similar level to CDA1. The CAN19 mAbsshowed little cross-reactivity to toxin B. CAN20 mAbs bind to toxin A.CAN20G2 and CAN20G5 bind to toxin A fragment 4 at a similar level toCDA1. None of CAN20 mAbs showed cross-reactivity to toxin B.

Example 4 Western Blot of Mouse Monoclonal Antibodies

A 4-12% SDS-PAGE gel was run for 1.5 hours at 200 volts with acombination of C. difficile proteins; whole toxin A, toxoid A(commercial), recombinant toxin fragment 4 and toxin B (whole, toxoidand fragment 4). The gel was then transferred to a nitrocellulosemembrane for 45 min at 45 volts. The membrane was blocked overnight at4° C. with 1% skim milk in 1×TBST and the next day washed with 1×TBST toremove the skim milk. The mAbs (1° Ab) were diluted in 1×TBST at aconcentration of 1 μg/ml and used to probe the membrane containing thetransferred products for 2 hours at room temperature (RT) on a shaker.The membranes were then washed with 1×TBST to remove unbound 1° Ab andprobed with anti-mouse IgG-HRP (2° Ab) at a dilution of 1:4000 for 1hour at RT on a shaker.

Results:

As shown in FIGS. 4 and 5, all three CAN19 mAbs showed binding to wholetoxin A, toxoid A and toxin A fragment 4. They all showed only weak orno cross-reactivity to toxin B or to the negative control. CAN20G1,CAN20G2, CAN20G5 and CAN20G8 mAbs all showed strong binding to wholetoxin A and toxin A fragment 4. There was no cross-reactivity to toxin Bor to the negative control.

Example 5 Affinity Analysis of Mouse Monoclonal Antibodies

Biolayer interferometry was used to measure the interactions betweenwhole Toxin A and the anti-toxin A antibodies. The Octet QKe instrument(ForteBio) was equipped with Streptavidin (SA) biosensors. 40 μg/ml ofbiotinylated whole Toxin A was coupled to SA sensors and the toxin AmAbs, in a dilution series from 100 nM to 1.56 nM, were reacted on thetoxin-coated pins for 10 minutes followed by a dissociation step in PBSfor another 10 minutes. The results were then analyzed using ForteBioData Analysis software to determine the dissociation constant (K_(D)),which is the measure used to describe the binding strength betweenantibody and antigen, k_(on)(1/Ms), the on-rate at which antibodyantigen complexes form, and k_(dis)(1/s), the off-rate at which theantibody antigen complexes dissociate. The samples were run over twoseparate days. Table 4 shows affinity data for purified CAN20G versions,as well as CDA1.

TABLE 4 Affinity data for purified CAN20G versions and CDA1 ID NameK_(D) (M) k_(on)(1/Ms) k_(dis) (1/s) CAN20G1 1.79E−09 1.23E+05 2.19E−04CAN20G2 4.19E−12 1.04E+05 4.35E−07 CAN20G5 2.01E−09 8.38E+04 1.68E−04CAN20G8 1.65E−09 1.31E+05 2.16E−04 CDA1 6.24E−10 4.80E+05 2.91E−04

Example 6 Epitope Binning of Mouse Monoclonal Antibodies

The Octet QKe is a label free real-time biosensor that uses disposablefiber-optic sensors that detect biomolecular interactions via biolayerinterferometry. The epitope binning assay was performed against thepreviously characterized CDA1 anti-toxin A mAb to examine whether thepresent toxin A mAbs share a similar or a different epitope with CDA-1.Secondly, the assay was used to confirm shared single or potentiallymultiple epitope bins between the toxin A mAbs. The classical sandwichmethod was used and involves coupling the mAb to sensor, bindingantigen, and then binding to another mAb. The second mAb can bind thecaptured Ag only if its epitope does not overlap that of the immobilizedmAb.

Results:

The strong nM shift in wavelength above the CAN20G1 and PBS control (avertical increase in the binding curve) indicates more binding is ableto occur and that the test antibody is binding to an exposed anddistinct epitope. As shown in FIGS. 6a and 6b , the results indicatethat there is an elevated shift in wavelength for the CDA1 antibody.This indicates that the CDA1 and CAN20 mAbs bind to distinct epitopes.All the CAN20 mAbs share the same epitope. There is a slight nmelevation for the CAN20G2 indicating a slight increase in binding whichcould be due to a somatic mutation between the known VH and VL chains ofCAN20G1 and CAN20G2, different antibody epitopes or both.

Example 7 In Vitro Neutralization of Mouse Monoclonal Antibodies

The in vitro neutralization assays described herein were performed usingVERO (green monkey) cells and Toxin A purchased from List BiologicalLaboratories. (BIAD report: Clostridium difficile Toxin A MonoclonalAntibody Characterization). The protocols used for xCelligence (RocheDiagnostics) and Bioassy methods are summarized below.

Cell Attachment Phase—xCelligence Method.

(1) Trypsinized cells in source flask. (2) Added 2 mL of trypsin toflask and washed cells to remove traces of media then aspirate. (3)Added 3 mL of trypsin and incubated at 37° C. for approximately 10minutes until cells were detached. (4) Added 6 mL of assay media orgrowth media to flask. (4) Centrifuged at 1300 rpm for 8 minutes. (5)Aspirated supernatant and resuspended cells with 6 mL of Assay media orgrowth media. (6) Counted cells and calculated required cell density.(For Vero cells, 1×10⁵ cells/mL and for T84 cells, 8×10⁵ cells/mL.) (7)To a 96 well E-plate added 100 μL of Assay media to wells A1 thru H10and 100 μL of T84 media to wells A11 thru H12. (8) Performed backgroundreading on xCelligence. (9) Removed 50 μL of Assay media from wellsA1-H10. (10) Added 50 μL of 1.0×10⁵ cells/mL suspension to these wellsfor a final 5.0×10⁴ cells/mL seeding density. (11) Added 100 μL of T848×10⁵ cells/mL cell suspension to A11 and A12. (12) Serially diluted2-fold down through H11 and H12. (13) Remove 100 μL from H11 and H12.(14) Added 100 μL of T84 media to A11-H12 for a final volume 200 μL.(15) Incubated plate at room temperature for 20-30 minutes to allowcells to settle evenly. (16) Placed plate in 37° C. incubator with 5%CO2 overlay for 20-24 hours.

Cell Attachment Phase—Bioassy Method.

(1) Trypsinized cells in source flasks. (2) Pooled cells from sourceflasks. (3) Centrifuged cells at 1270 RPM for 8 minutes. (4) Removedsupernatant and resuspended cells in assay medium. (5) Six mL of mediumshould be used for every flask pooled. (6) Counted cells to determinecell viability and quantity of cells required to plate at 1.0×10⁵cells/mL. (7) Final concentration will be 0.5×10⁵ cells/mL when plated.(8) Added 50 μL of 10% Assay Media to wells B2-G11 of a 96 well blackclear-bottom microplate. (9) Overlayed 50 μL of cells to wells B2-G11 ofa 96 well flat-bottom microplate at 1.0×10⁵ cells/mL. (10) To the outeredge wells, added 100 μL of warmed assay media. (11) Mixed on a plateshaker for a homogeneous suspension. (12) Left plate at room temperaturefor 20-30 minutes to allow cells to settle evenly across the wells. (13)Placed cell plates in a 37° C., 5% CO₂ humidified incubator for 20-24hours.

Toxin a Preparation:

(1) Prepared Toxin A primary stock (20 μg/mL) by adding 100 μL ofsterile LW to one vial (2.0 μg) of Toxin A. (2) Diluted primary stock asshown in Table 5.

TABLE 5 TcdA Final Plating Volume of TcdA Concentration ConcentrationPrimary Stock Volume of 10% (ng/mL) (ng/mL) (20 μg/mL) Assay Medium 6020 12 μL 3988 μL

Sample Preparation:

To test potency, all the monoclonal antibodies were at a startingconcentration of 30 μg/mL. Samples were prepared as shown in Table 6.

TABLE 6 Sample (Stock Preparation Final Plating Volume Volume Assayconcentration) Concentration Concentration Sample Stock Medium CDA(1.556 mg/mL) 30 μg/mL 10 μg/mL 28.9 μL 1471.1 μL CAN19 G1 30 μg/mL 10μg/mL  150 μL/plate n/a Purified 30 μg/mL CAN19 G2 30 μg/mL 10 μg/mL 150 μL/plate n/a Purified 30 μg/mL CAN19 G3 30 μg/mL 10 μg/mL  150μL/plate n/a Purified 30 μg/mL

Dilution Plate Preparation—xCelligence:

(1) Added assay media and 150 μL of sample to wells as shown below inTable 7. (2) Serially diluted each sample 2-fold down the column bytaking 75 μL from Row A and adding to Row B, mixed 3 to five times andrepeated down through to Row G. (3) Added appropriate controls to wellsas shown in Table 5. (4) Overlayed sample wells with 75 μL of Toxin A.Shake on a plate shaker until homogeneous. (5) Incubated at 37° C. for 1hour.

TABLE 7 xCelligence Dilution Plate Layout Cell Density: 0.5e5 cells/mLCDA CDA G1 G1 G2 G2 G3 G3 G1 Sup G1 Sup G2 Sup G2 Sup 1 2 3 4 5 6 7 8 910 11 12 A Sample Sample Sample Sample Sample Sample CDA Ctl CDA CtlSample Sample 150 uL 150 uL B 75 uL 75 uL 75 uL 75 uL 75 uL 75 uL 75 uL75 uL 75 uL 75 uL 150 uL 150 uL C 75 uL 75 uL 75 uL 75 uL 75 uL 75 uL 75uL 75 uL 75 uL 75 uL 150 uL 150 uL D 75 uL 75 uL 75 uL 75 uL 75 uL 75 uL75 uL 75 uL 75 uL 75 uL 150 uL 150 uL E 75 uL 75 uL 75 uL 75 uL 75 uL 75uL 75 uL 75 uL 75 uL 75 uL 150 uL 150 uL F 75 uL 75 uL 75 uL 75 uL 75 uL75 uL 75 uL 75 uL 75 uL 75 uL 150 uL 150 uL G 75 uL 75 uL 75 uL 75 uL 75uL 75 uL 75 uL 75 uL Cell Ctl Cell Ctl Tox Ctl Tox Ctl H CDA Ctl CDA CtlG1 Ctl G1 Ctl G2 Ctl G2 Ctl G3 Ctl G3 Ctl G1 Sup Ct

G1 Sup Ct

G2 Sup Ct

G2 Sup Ct

Cell control = 150 μL AM 75 uL/150 uL = volume AM Toxin control = 75 μLtoxin + 75 μL AM mAb control = 75 μL mAb + 75 μL AM Sample = 150 uLrespective sample

indicates data missing or illegible when filed

Bioassy Method:

Added assay medium to wells as shown in Table 8. (1) Add 150 μL ofsample to appropriate wells of column 2. On plate #1, add CDA, CAN19G1(purified), and CAN19G1 supernatant. On plate #2 added CDA, CAN19G2purified, and CAN19G2 supernatant. On plate #3 added CDA, CAN19G3purified, and CAN19G3 supernatant. (2) Transferred 75 μL from column 2to column 3. Mixed with multichannel. Repeated procedure through tocolumn 9. (3) Remove 75 μL from column 10 leaving a final volume 100 μL.(4) Added controls (75 μL) to appropriate wells along with 75 μL of AM.(5) Overlayed sample wells with 75 μL of Toxin A.

TABLE 8 Bioasssay Dilution Plate Layout Cell Density: 0.5e5 cells/mL 50μL Serial Dilutions 1 2 3 4 5 6 A 200 μL A

200 μL A

  200 μL A

  200 μL A

  200 μL A

  200 μL A

  B 200 μL A

CDA 75 μL AM 75 μL AM 75 μL AM 75 μL AM C 200 μL A

CNJ mAb 75 μL AM 75 μL AM 75 μL AM 75 μL AM D 200 μL A

CNJ sup 75 μL AM 75 μL AM 75 μL AM 75 μL AM E 200 μL A

CDA 75 μL AM 75 μL AM 75 μL AM 75 μL AM F 200 μL A

CNJ mAb 75 μL AM 75 μL AM 75 μL AM 75 μL AM G 200 μL A

CNJ sup 75 μL AM 75 μL AM 75 μL AM 75 μL AM H 200 μL A

200 μL AM  200 μL A

  200 μL A

  200 μL A

  200 μL A

  Cell control = 150 μL AM Toxin control = 75 μL toxin + 75 μL AM mAbcontrol = 75 μL mAb + 75 μL AM 50 μL Serial Dilutions Controls 7 8 9 1011 12 A 200 μL A

  200 μL A

  200 μL A

  200 μL A

  200 μL A

200 μL A

B 75 μL AM 75 μL AM 75 μL AM CDA ctl Cell 200 μL A

C 75 μL AM 75 μL AM 75 μL AM mAb Ctl Toxin 200 μL A

D 75 μL AM 75 μL AM 75 μL AM sub Ctl Cell 200 μL A

E 75 μL AM 75 μL AM 75 μL AM CDA ctl Toxin 200 μL A

F 75 μL AM 75 μL AM 75 μL AM mAb Ctl mAb 200 μL A

G 75 μL AM 75 μL AM 75 μL AM sub Ctl pAb 200 μL A

H 200 μL A

  200 μL A

  200 μL A

  200 μL A

  200 μL A

200 μL A

indicates data missing or illegible when filed

Sample Addition to Cell Plates:

(1) Following 1 hour incubation, cell plates were removed fromincubator. (2) Removed 50 μL of cell suspension carefully withmultichannel pipette being sure not to disturb cell monolayer. (3)Transferred 100 μL of samples from dilution plate to appropriate wellsof cell plate. (4) Mixed on plate shaker for a homogeneous solution.Incubated 72 hours at 37° C. with a 5% CO2 overlay.

Data Analysis:

The xCelligence system captures data in real-time. For the purposes ofcomparison to the conventional bioassay methods, the final read timedata is analyzed. For this, we normalized the cell index at the timepoint before toxin/antibody addition to the plate, using the appropriatetoxin wells as baseline. This will create a baseline normalized cellindex on the Y axis versus log concentration of antibody. We analyzedthe data to determine potency of CAN19 mAbs in comparison to CDA. %Neutralization is calculated as follows with xCelligence:

% Neutralization=(Sample CI index/Antibody Control CI index)*100%

Neutralization is calculated as follows with Bioassay fluorescence:

% Neutralization=(Mean Sample RFU/Mean Toxin RFU)/(Mean Cell RFU/MeanToxin RFU)*100

The procedures of this Example were also performed on CAN20 mAbs.

Results:

CAN19 mAbs were less neutralizing than CDA1. CAN20G2 is the most potentmAb in vitro and is more potent than CDA1. CAN20G3, G5 and G8 are alsoneutralizing.

Table 9 summarizes the IC₅₀ data generated for each CAN19 mAbdemonstrating that the CAN19 clones are less neutralizing compared toCDA1.

TABLE 9 Sample IC50 CDA 0.347 ug/mL  standard 1 2.31 ug/mL CAN19G1 22.17 ug/mL CAN19G2 3 2.44 ug/mL CAN19G3

Table 10 summarizes the EC₅₀ data generated for each CAN20 mAbdemonstrating that CAN20G2, CAN20G3, CAN20G5, and CAN20G8 are the mostneutralizing of the clones.

TABLE 10 Calculated anti- TcdA IgG Concentration By EC50 Value ID NameBiacore(μg/mL) (μg/mL)¹ CAN20G1 188.0 0.17 CAN20G2 142.2 0.0101 CAN20G35.9 0.076 CAN20G4 22.8 0.147 CAN20G5 87.5 0.13 CAN20G6 314.2 0.151CAN20G7 134.9 0.158 CAN20G8 272.4 0.137 ¹The EC50 value is theconcentration of antibody which neutralizes 50% of the TcdA toxin dose.

Example 8 Mouse In Vivo Toxin Challenge

The mouse in vivo toxin challenge test was based on previouspublications (Babcock et al., Human Monoclonal Antibodies Directedagainst Toxins A and B prevent Clostridium difficile-Induced Mortalityin Hamsters. Infection and Immunity (2006) 74(11):6339). Swiss webstermice weighing 20-30 g were given 250 μg of mAb or controls at day 0 andallowed to rest. After 24 hrs (day 1), the mice were given a lethal doseof TcdA (100 ng). This dose kills 90-100% of animals by 24 hours in anunprotected state. The mice were observed for 7 days (days 1-7) forsigns of abnormality and local and systemic disease. The mice wereeuthanized on Day 7. All observations were recorded and the % survivalwas determined for each treatment group.

Results:

As shown in FIGS. 7, 8, and 9, the study results indicate that the CAN19and CAN20 mAbs protect mice against toxin A. There was >90% survivalwith CAN19G1, G2 and G3. All three CAN19 mAbs showed efficacy. All theCAN20 mAbs were efficacious. CAN20G1, G2, G5 and G8 showed 100%protection at a dose of 0.25 mg/mouse. CAN20G2 showed 100% protection at0.125 mg/mouse. The experiment was repeated to confirm the efficacy ofCAN20G2. The results confirmed the previous study. CAN20G2 showed 100%protection at the full does of 0.25 mg/mouse and 90% protection at thehalf dose 0.125 mg/mouse.

Example 9 muCAN20G2 V Gene Sequencing

RNA was isolated from the CAN20G2 parental hybridoma clonal cell lineusing the RNeasy Mini Kit. The amplification of V genes from the RNA wasperformed using the Qiagen OneStep RT-PCR Kit. Several combinations ofprimer sets were used as follows: for immunoglobulin variable regiongene sequence confirmation from the hybridomas, a set of Variable regiongene (V-gene) subgroup-specific oligonucleotide primers are used. Theseinclude 5′mVK-Lead-1, 3′KappaConstRT, 5′mVH-Lead-2, 5′mVH-Lead-2A, and3′mIG1-2C RT. In order to rule out potential contamination from theknown and endogenous aberrant kappa light chain V-gene mRNA (foundwithin P3X63 myelomas) (Yuan, X. et al., J. Immunol. Methods, 294:199-207 (2004)), the RT-PCR was also performed using non-subgroupspecific primer sets, 5′mVK-Lead-1A, 5′mVK-Lead-1A, 5′mVK-Lead-3,5′mVK-Lead-3A, 5′mVH-IGHV1-Lead, 5′mVH-Lead-1, 5′mVH-Lead-3,5′mVH-Lead-4, and 5′mVH-Lead-5. Refer to FIG. 10 for a list of theprimers and their sequences. The results of the PCR amplificationreactions were determined by examining the PCR products on an analyticalagarose gel, and the visualized bands at approximately 500 bp were gelisolated for cloning. The extracted DNA was directly TA cloned into thepCR2.1-TOPO vector using the low melt agarose method in the TOPO TACloning manual. Five colonies of each CAN20G clone reaction weresequenced in both directions using the M13 Forward and M13 Reverseprimers. Sequence data was analyzed using DNAStar Lasergene software.The resulting rearranged V-gene sequences were compared to IMGT/V-Questreference directory sets and to the NCBI immunoglobulin blast search(FIG. 11).

Example 10 Humanization of muCAN20G2

Three humanized IgG/k versions of CAN20G2 mAb have been created as wellas a chimeric IgG1/k. For the humanized versions, maximum identityalignment with human germline alleles was used (from the IMGT and NCBIwebsites) to help to identify acceptor frameworks. All 6 CDRs wereinserted. Other residues were changed or maintained due to surfaceexposure or involvement in folding or interchain contacts, respectively.The CDRs of the murine mAb sequence (CAN20G2) match very well with thegermline CDRs of the closest human alleles. This resembles the“superhumanization” approach where CDR matching rather than totalframework is used in a variation of the use of germline sequences asacceptor frameworks. In the case of Tan et al., J. Immunol. 2002,169:1119-1125, the authors used the CDR sequences and tried to match theso called canonical classes of CDRs based upon the Chothiaclassification system. However, because particular CDRs are germlineencoded and particular canonical conformations tend to be found incertain frameworks, the “Superhumanization” method of choosing acceptorframeworks does not in all cases result in the selection of a differentcandidate acceptor framework. It is empirical and remains to be testedfor multiple mAb specificities. This is in part because the straight-upalignment of frameworks for identity inherently encompasses the CDRs aswell in the comparison. Table 11 shows the percent humanness, at theamino acid level, of each of the humanized constructs of CAN20G2.

TABLE 11 PERCENT CONSTRUCT “HUMAN” MURINE CAN20G2 66% CHIMERIC CAN20G290% HE-CAN20G2 91% hCDR-CAN20G2 97% AVA-CAN20G2 95%FIG. 12 shows the alignment of muCAN20G2 v-regions with the closesthuman germline v-region. The human germlines were used as acceptorframeworks for humanization.

CDR-huCAN20G2—CDR Grafted Only.

The best matching germline allele for both V_(H) and Vk were used as anacceptor framework for grafting the CDRs. No other changes were made tothe acceptor frameworks. FIGS. 13a and 13b show the design of theCDR-huCAN20G2 design we used. The closest matching human frameworks areIGHV7-4-1*02 and IGKV1-39*01. The CDRs (IMGT Numbering) of the muCAN20G2were inserted into the human framework. The heavy CDR3 contained a HpaIrestriction site that was altered for cloning into pcDNA3002Neo. A 5′Kozak and HAVT20 leader sequence was added for correct translation andtrafficking.

HE-huCAN20G2 “Human Engineered”

This humanized version was generated using a strategy most similar tothe “human engineering” strategy used by Studnicka et al (1994) used tohumanize a murine mAb to CD5. Essentially, the closest human germlineallele for both CAN20G2 V_(H) and Vk were identified, individually, anddesigned for use as acceptor frameworks. The CAN20G2 V_(H) has a 76%identity with the human IgVH7-4-1*02 allele. The CDRs were grafted oraltered to match the CAN20G2 mAb sequences. The HE-hCAN20G2 antibodiesare shown in FIGS. 14, 15, and 16. Some residues were modified ormaintained as described in the legend. In this case, crystal structuralinference was taken from Avastin/Bevacizumab. Avastin is a humanizedmonoclonal antibody that recognizes and blocks vascular endothelialgrowth factor A (VEGF-A) and is marketed for the treatment of advancedcolorectal cancer. Avastin turns out to have highest identity with thesame human germline gene as CAN20G2 VH and the crystal structure of itsvariable region structure has been determined.

AVA-huCAN20G2 “Avastinized”—

Alignment of the translation of the Avastin V_(H) and Vκ/Jκ alleles withthe respective humanized CAN20G2 V_(H) and Vκ immunoglobulin variableregions is shown in FIG. 17. Many mAbs have been humanized capitalizingon the natural sequence pairing of V_(H) and V_(L) found in other mAbswith crystal structural data. In this case, we used the same V_(H) as inVersion 1-HE (which has high identity with Avastin V_(H)), and we usedthe Avastin Vk as the Light chain acceptor framework. This allowed us toexploit the known interchain contacts and modification in our design(FIG. 18).

Chimeric-huCAN20G2 Chimeric Version:

A chimeric CAN20G2 was designed as a control. Certain residues outsidethe CDRs are involved in the structure of the hypervariable regions.During the humanization process some of the residues may be altered.Because sequence variation within the canonical structures will modulatethe conformation of the paratope, it is essential to determine whetherthe loss/gain in affinity/function/neutralization is due to thehumanization process or the human Fc region. The CAN20G2 murinev-regions were designed onto human IgG1 and human Kappa constantregions. The construct contains Kozak, HAVT20 Leader and double stopsequences (FIGS. 19A and 19B).

Example 11 SDS Page and Western Blot Analyses of Humanized Antibodies

A large scale transfection (300 ml) was performed in HEK293F cells toobtain a large quantity of each huCAN20G2 mAb. A total of 3×10⁸ cellswere transfected with 300 μg of huCAN20G2 plasmid DNA. The supernatantwas harvested by centrifugation (3000 rpm, 15 min, RT) 3 days and 7 dayspost-transfection. The transfected supernatant was filtered through a0.22 μm filter. The filtered sup was purified on a Protein G column(HiTRAP HP, GE Healthcare) using the AktaPurifier FPLC. The elutedprotein was buffer exchanged into D-PBS and the concentration determinedby BCA assay. A range of 30-45 mg was purified from the 300 ml cultures.The purified protein was run on an SDS-page to confirm its size (FIG.22). The purified mAb was also used to probe a membrane with whole toxinA and toxin A fragment 4 to confirm the binding characteristics of themAbs (FIG. 23).

Example 12 In Vitro Neutralization Assay of Humanized Antibodies

An in vitro neutralization assay for C. difficile Toxins using CT-26cells was performed to test the neutralization capability of thehumanized mAb clones against C. difficile toxin A. The CT-26 cells wereseeded in a 96 well plate at a concentration of 2.5-3×10⁴ cells/100ul/well and the plate was incubated in a CO₂ incubator for 4-5 hrs at37° C. Two blank wells containing only media (no cells) were alsoincluded in the plate.

The toxin and toxin/Ab mixtures were prepared in tubes and diluted tothe desired concentrations using RPMI media. The tubes were left toincubate at room temperature for 1 hour. The media was removed from thewells of the plate and each of the tubes, containing either media alone,toxin alone, or toxin/Ab mixtures, was transferred to its designatedwell. The plates were left to incubate for 48 hours at 37° C. and 5%CO₂. The WST-1 detection reagent was added to each well (10 μl ofreagent/100 μl volume in the well) and incubated for 1 hour at 37° C.and 5% CO₂. The plate was shaken for 1 min and then read at 450 nm.

Cell Viability was Determined Based on the Cell Controls as Below:

% Cell viability=Mean OD of test/Mean OD of cell control×100.

Toxin neutralization is calculated by the formula as below:

% Neutralization=(Sample OD−Toxin control OD)/(Cell control OD−toxincontrol OD)*100

Results:

As shown in FIGS. 20a and 20b , the chimeric CAN20G2 and the HE-CAN20G2are the most neutralizing at all mAb concentrations. The HE-CAN20G2 ismore neutralizing at most mAb concentrations at either Toxin Aconcentration. The Medarex CDA IgG and the hCDR mAbs show similar modestneutralization ability and the AVA-CAN20G2 shows very littleneutralization ability.

Example 13 Affinity Assay of Humanized Antibodies

Biolayer interferometry was used to measure the interactions betweenwhole Toxin A and the humanized CAN20G2 antibodies. The Octet QKeinstrument was equipped with Streptavidin (SA) biosensors. 40 μg/ml ofbiotinylated whole Toxin A was coupled to SA sensors and the humanizedversions, in a dilution series from 100 nM to 1.56 nM, was allowed toassociate with the toxin for 10 minutes followed by a dissociation stepin PBS for another 10 minutes. The results were then analyzed usingForteBio Data Analysis software to determine K_(D) (nM), the measureused to describe the binding strength between antibody and antigen,k_(on)(1/Ms), the rate at which antibody antigen complexes form, andk_(dis)(1/s), the rate at which the antibody antigen complexesdissociate.

Results:

The results from two experiments were averaged and show that themuCAN20G2 and the chCAN20G2 are within threefold indicating no loss inaffinity (Table 10). In contrast, the AVA-CAN20G2 showed almost a fulllog loss in affinity. The CDR-huCAN20G2 showed loss in affinity nearingthat of the AVA humanized version. The binding affinity of theHE-huCAN20G2 version is slightly higher than all the other humanizedversions but within the acceptable threefold range showing little or noloss of affinity compared to the chimeric CAN20G2. We believe this isthe optimal comparator because we cannot predict the effects ofexchanging the human constant regions for the murine IgG2a constantregions and this comparison takes this into account. The three foldrange comparison is considered by the ForteBio experts as insignificantvariation.

TABLE 12 Affinity data for purified human CAN20G2 versions. K_(D) (M)k_(on) (1/Ms) k_(dis) (1/s) muCAN20G2-2-1 1.66E−10 1.08E+05 1.80E−05chCAN20G2 1.72E−10 1.14E+05 1.93E−05 AVA-CAN20G2 1.33E−09 5.45E+049.02E−05 HE-huCAN20G2 3.32E−10 9.39E+04 3.14E−05 CDR-huCAN20G2 8.00E−106.76E+04 5.41E−05

Example 14 ELISA Testing of Humanized Antibodies

A medium scale (150 ml) transfection was performed in HEK293F cells totest for expression of the huCAN20G2 mAb. A total of 1.5×10⁸ cells weretransfected with 150 μg of DNA. The supernatant was harvested bycentrifugation (3000 rpm, 15 min, RT) 3 days and 7 dayspost-transfection. The transfected supernatant was filtered through a0.22 μm filter. The filtered supernatant from the medium scaletransfection was screened with an ELISA prior to purification. An ELISAwas run to test the binding of the human mAb clones against whole toxinA and toxin A fragment 4. The human mAb clones were compared to CDA1 andthe chimeric CAN20G2. The ELISA plate was coated with 100 μg/ml of ToxinA fragment 4 and 400 μg/ml of whole Toxin A so that the coats wereequimolar. The coats were probed with serially diluted mAb (0.128 ng/mlto 10 μg/ml) and binding was detected with anti-human IgG-HRP antibody.The plate was read at 405 nm after 60 min incubation with substrate.

Results:

As shown in FIGS. 21 a-d, all three humanized versions of mAb CAN20G2,in addition to the chimeric version, bind to whole toxin A with similarintensity in ELISA. In contrast, there are clearly differences in thebinding of the humanized mAbs to recombinant toxin A fragment 4, whichis the domain of Tcd A to which the parental CAN20G2 is known to map andbind. This may be indicative of the functionality if this binding tofragment 4 correlates with in vitro and in vivo protection and may allowthe development of domain 4 assays as a surrogate for CAN20G2 efficacy.The chimeric and HE mAbs appear to bind similarly whereas the CDR mAbbinds to a lesser degree and the AVA mAb does not appear to bind to thetoxin A fragment 4.

Example 15 In Vivo Challenge with Tcd A

Based on the in vitro data, the CDR and HE humanized versions of CAN20G2were tested in vivo and compared to the chimeric version in the mouselethal toxin challenge model (as noted in Example 8 above). SwissWebster mice weighing 20-30 g were given 250 ug of mAb or controls atday 0 and allowed to rest. After 24 hours, the mice were administered alethal dose of TcdA (100 ng). This dose kills 100% of animals by 24hours in an unprotected state. The mice were observed for a period of 4days for clinical symptoms, abnormality and local and systemic disease.All observations were recorded and the results summarized in Table 13which shows all the antibodies tested, including the HE and CDR versionsare effective at neutralizing toxin A and protecting against toxin Achallenge in vivo.

TABLE 13 Effect of Can20G2 humanized MAbs against Tcd A challenge inmice. # Dead/euthanized Groups Treatment N # Survivors during the studyA chimeric-Can20G2 5 5 0 B HE-Can20G2 5 5 0 C hCDR-Can20G2 5 5 0 DmuCan20G2 5 5 0 E CDA1-1 5 5 0 F Rb-polyclonal 5 5 0 G TcdA/PBS controls5 0 5 H PBS alone 4 4 0

Example 16 Immunogenicity Analysis of Humanized Antibodies

In order to determine their immunogenicity, CDR-huCAN20G2 andHE-huCAN20G2 were tested in the EpiScreen™ (Antitope Ltd) time course Tcell assays, using two markers (proliferation and IL-2 production) tomeasure T cell activation. Specifically, peripheral blood mononuclearcells (PBMCs) were prepared from a cohort of 21 healthy donors withrepresenting HLA (Human Leukocyte Antigen) allotypes. Bulk cultures wereestablished using CD8⁺-depleted PBMCs. CD4⁺ T cell proliferation byincorporation of [³H]-Thymidine was measured at various time pointsafter the addition of the antibodies. IL-2 secretion was also measuredusing ELISpot assays in parallel to the proliferation analysis.

Methods Preparation and Selection of Donor PBMCs

Peripheral blood mononuclear cells (PBMCs) were isolated from healthycommunity donor buffy coats (from blood drawn within 24 hours). PBMCswere isolated from buffy coats by Lymphoprep (Axis-shield, Dundee, UK)density centrifugation and CD8⁺ T cells were depleted using CD8⁺RosetteSep™ (StemCell Technologies Inc, London, UK). Donors werecharacterized by identifying HLA-DR haplotypes using an HLA SSP-PCRbased tissue-typing kit (Biotest, Solihull, UK). T cell responses to acontrol antigen (Keyhole Limpet Haemocyanin (KLH), [Pierce (Perbio),Cramlington, UK]), as well as peptides derived from Influenza A andEpstein Barr viruses were also determined. PBMCs were then frozen andstored in liquid nitrogen until required.

Preparation of Antibodies

The two test antibodies were diluted in AIM-V® culture medium(Invitrogen, Paisley, UK) just before use and the final assayconcentration was 0.3 mM. KLH was used as a reproducibility control andstored at −20° C. as a 10 mg/ml stock solution in water. For thestudies, an aliquot of KLH was thawed before immediately diluting to 400μm/ml in AIM-V® (final concentration 100 μm/ml). Phytohaemagglutanin(PHA, Sigma, Poole, UK) was used as a positive control in the ELISpotand a 1 mg/ml stock was stored at −20° C. before diluting to a finalconcentration of 2.5 μm/ml in cell cultures.

Assessment of Cell Viability

On day 7, bulk cultures (previously established for the proliferationassay) were gently resuspended and 10 ml of each sample was removed fromall donors and mixed with 10 ml trypan blue. These samples were thenassessed for viability using trypan blue dye exclusion with a Countess®Automated Cell Counter instrument (Invitrogen).

EpiScreen™ Time Course T Cell Proliferation Assays

PBMCs from each donor were thawed, counted and viability assessed. Cellswere revived in room temperature AIM-V® culture medium, washed andresuspended in AIM-V® to 4-6×10⁶ PBMC/ml. For each donor, bulk cultureswere established in which 1 ml proliferation cell stock was added to theappropriate wells of a 24 well plate. 0.5 ml of culture medium and 0.5ml of each diluted antibody were added to the PBMC to give a finalconcentration of 0.304. For each donor, a reproducibility control (cellsincubated with 100 μg/ml KLH), a positive control (cells incubated with2.5 μg/ml PHA) and a culture medium-only well were also included.Cultures were incubated for a total of 8 days at 37° C. with 5% CO₂. Ondays 5, 6, 7 and 8, the cells in each well were gently resuspended and3×100 μl aliquots transferred to each well of a round bottomed 96 wellplate. The cultures were pulsed with 0.75 μCi [³H]-Thymidine (PerkinElmerR, Beaconsfield, UK) in 100 μl AIM-VR culture medium and incubatedfor a further 18 hours before harvesting onto filter mats (PerkinElmerR) using a Skatron Micro 96S-10056 cell harvester. Counts perminute (cpm) for each well were determined by Meltilex™ (Perkin ElmerR)scintillation counting on a 1450 Microbeta Wallac Trilux LiquidScintillation Counter (Perkin ElmerR) in paralux, low backgroundcounting.

EpiScreen™ IL-2 ELISpot Assays

Homologous donors to those used in the proliferation assay were alsoused for the IL-2 ELISpot assay. Cells were thawed and revived asdescribed above. ELISpot plates (Millipore, Watford, UK) were pre-wettedand coated overnight with 100 μl/well IL-2 capture antibody (R&DSystems, Abingdon, UK) in PBS. Plates were then washed 3 times in PBS,incubated overnight in blocking buffer (1% BSA in PBS) and washed inAIM-V® medium. The cell density for each donor was adjusted to 4-6×10⁶PBMC/ml in AIM-V® culture medium and 100 μl of cells were added to eachwell. 50 μl of samples and controls were added to the appropriate wellsas well as 50 ml of AIMV to bring the total volume to 200 ml/well.Antibodies were tested in sextuplicate cultures and, for each donor, anegative control (AIM-V® medium alone), no cells control and a mitogenpositive control (PHA at 2.5 μg/ml—used as an internal test for ELISpotfunction and cell viability), were also included on each plate. After an8 day incubation period, ELISpot plates were developed by sequentialwashing in dH₂O and PBS (×3) prior to the addition of 100 μl filtered,biotinylated detection antibody (R&D Systems) in PBS/1% BSA. Followingincubation at 37° C. for 1.5 hours, plates were further washed in PBS(×3) and 100 μl filtered streptavidin-AP (R&D Systems) in PBS/1% BSA wasadded for 1.5 hours (incubation at room temperature). Streptavidin-APwas discarded and plates were washed in PBS (×4). 100 μl BCIP/NBTsubstrate (R&D Systems) was added to each well and incubated for 30minutes at room temperature. Spot development was stopped by washing thewells and the backs of the wells three times with dH₂O. Dried plateswere scanned on an Immunoscan® Analyser and spots per well (spw) weredetermined using Immunoscan® Version 4 software.

EpiScreen™ Data Analysis

For proliferation and IL-2 ELISpot assays, an empirical threshold of astimulation index (SI) equal to or greater than 2 (SI≧2.00) has beenpreviously established, whereby samples inducing responses above thisthreshold are deemed positive (borderline SIs≧1.90 are alsohighlighted). Extensive assay development and previous studies haveshown that this is the minimum signal-to-noise threshold allowingmaximum sensitivity without detecting large numbers of false positiveresponses or omitting subtle immunogenic events. For both proliferation(n=3) and IL-2 ELISpot data (n=6) sets, positive responses were definedby statistical and empirical thresholds as follows:

-   -   1. Significance (p<0.05) of the response by comparing cpm or spw        of test wells against medium control wells using unpaired two        sample student's t-test.    -   2. Stimulation index greater than or equal to 2 (SI≧2.00), where        SI=mean of test wells (cpm or spw)/baseline (cpm or spw). Data        presented in this way is indicated as SI≧2.00, p<0.05.        In addition, intra-assay variation was assessed by calculating        the coefficient of variance and standard deviation (SD) of the        raw data from replicate cultures.

Results & Discussion

While there is generally a good correlation between IL-2 production andproliferation after T cells have been activated, proliferation and IL-2ELISpot assays have been interpreted independently. Inter-assayvariability was assessed using KLH as a reproducibility control wherethe frequency of positive T cell responses against KLH were compared intwo separate EpiScreen™ assays. The results show that interassayvariability for KLH-specific T cell responses is within the acceptablerange and consistent with previous studies (≦10%).

Assessment of Cell Viability

An initial assessment of any gross effect of the antibodies and thebuffer on PBMC viability was performed for 10 donors used in theEpiScreen™ time course assays. Cell viabilities were calculated usingtrypan blue dye exclusion of PBMC 7 days after culture with theantibodies. It was clear that the two test antibodies and bufferformulation did not significantly affect the viability of the cellsbecause PBMC from medium alone cultures had a mean viability similar tothat of the samples and KLH treated cells (between 93-97%).

EpiScreen™ Time Course Proliferation Assay

FIG. 24 and Table 12 show the results obtained in the EpiScreen™ timecourse T cell proliferation assay of CD4+ T cell responses induced bythe antibodies. Both test antibodies induced positive proliferationresponses with SI≧2.00 (p<0.05) in one or more donors in theproliferation assay. Borderline responses SI≧1.90 (p<0.05) are alsohighlighted. Positive proliferation responses ranged between 5% and 24%of the donor cohort (Table 14).

TABLE 14 Summary of T cell proliferation and IL-2 ELISpot responsesCDR-hu HE-hu Donor CAN20G2 CAN20G2 Buffer KLH Donor 1 PE Donor 2 PEDonor 3 PE PE PE Donor 4 PE* PE Donor 5 PE Donor 6 PE Donor 7 PE‡ EDonor 8 PE Donor 9 PE Donor 10 PE Donor 11 N/A PE Donor 12 N/A PE Donor13 N/A PE Donor 14 PE N/A E Donor 15 N/A PE Donor 16 PE N/A E Donor 17N/A PE Donor 18 N/A P Donor 19 N/A PE Donor 20 N/A PE Donor 21 N/A PEProliferation % 24 5 0 86 ELISpot % 24 5 0 95 Proliferation 24 5 0 81and ELISpot % Correlation % 100 100 N/A 94In Table 14, during the entire time course (days 5-8), positive T cellproliferation responses (SI≧2.00, significant p<0.05) were indicated as“P”, and positive T cell IL-2 ELISpot responses (SI≧2.00, significantp<0.05) were indicated as “E”. Borderline responses (significant p<0.05with SI≧1.90) was shown as (*). No data was obtained on day 8 of theproliferation assay for donor 7 (‡). Formulation buffer was tested ondonors 1-10 only donor 11-21 were not tested with the buffer (greyboxes). N/A indicated no data is available.

Antibody CDR-HuCAN20G2 was associated with the most frequent T cellproliferation response, inducing positive responses in 24% (5 donors) ofthe study cohort. In contrast, antibody HE-HuCAN20G2 induced fewer Tcell proliferation responses with only 5% of the cohort respondingpositively. These results showed that the frequency of T cellproliferation responses is high for antibody CDR-HuCAN20G2 but low forHE-HuCAN20G2. No T cell proliferation responses were detected againstthe buffer control.

Analysis of the magnitude of T cell proliferation responses showed thatalthough antibody CDR-HuCAN20G2 had a high frequency of response, themagnitude of responses were low (mean SI 2.13). For antibodyHE-HuCAN20G2 no conclusions can be made regarding the magnitude of the Tcell response due to the low number of responding donors (Table 15).Thus, the overall immunogenic potential of the antibodies was determinedbased on the frequency (%) of the positive T cell proliferationresponses in the study cohort with CDR-HuCAN20G2 being more immunogenicthan HE-HuCAN20G2.

TABLE 15 Summary of the mean magnitude (±SD) of positive T cellproliferation responses against the antibodies. Mean Frequency (%) ofSample SI +/− SD Response CDR-HuCAN20G2 2.13 0.09 24 HE-HuCAN20G2 2.250.13 5 KLH 2.60 0.78 86The mean SI was calculated from the average of all positive donorresponses observed during the entire time course (days 5-8). The dataincludes borderline proliferation responses (SI≧1.90, p<0.05).

Kinetics of T Cell Responses

The overall timing of the proliferative responses can provideinformation as to the potential type of T cell response (naïve orrecall). Maximal T cell proliferation detected on day 5 indicates thatexisting T cell precursor frequencies are high, whereas maximalproliferation on later days indicates a low existing T cell precursorfrequency. A high immunogenic potential would be concordant withstimulation of T cells during the early phase of the time course. FIG.25 summarizes the number of positive proliferation responses occurringagainst the samples on each day of the four day time course. The T cellresponses against antibody CDR-HuCAN20G2 were observed mostly on days 7and 8, suggesting that for this antibody the number of existing T cellprecursors is low. Antibody HE-HuCAN20G2 induced one donor to respondand this was observed on days 6, 7 and 8. However, since only oneresponding donor was detected it is difficult to make a conclusion as tothe number of T cell precursors for antibody HE-HUCAN20G2.

EpiScreen™ IL-2 ELISpot Assay

FIG. 26 and Table 12 show the responses obtained in the IL-2 ELISpotassay which measures IL-2 secretion by CD4+ T cells followingstimulation with the two test antibodies. Similar to the proliferationassay, positive responses were recorded in donors that produced anSI≧2.00 with a significant (p<0.05) difference observed between test spwand background (untreated medium control). Borderline responses SI≧1.90(p<0.05) are also highlighted. All samples induced positive IL-2 ELISpotresponses in one or more donors and these were all significant (p<0.05)using an unpaired, two sample student's t-test. All PHA wells werepositive for the presence of spots although SI values were not preparedfor the ELISpot data as, after 8 days, the majority of wells containedspots too numerous to count (data not shown).

For the two test antibodies, the overall results of the IL-2 ELISpotassay were homologous to those obtained in the proliferation assay withboth antibodies inducing the same frequency of T cell responses (Table16). As in the proliferation assay, antibody CDR-HuCAN20G2 induced themost frequent T cell responses in the study cohort with 24% of donorsresponding positively (SI≧2.00, p<0.05), whereas antibody HE-HuCAN20G2induced T cell responses in 5% of the study cohort. Assessment of themean magnitude of positive (including borderline SI≧1.90, p<0.05) T cellresponses against both antibodies was low (mean positive SI 2.39 forCDR-HuCAN20G2).

The frequency of T cell responses was low for HE-HuCAN20G2 whichprecludes making any direct correlation between strength of T cellresponse (magnitude) and immunogenicity. Assessment of the relative riskof immunogenicity of the test antibodies (based on the frequency ofpositive responses in the IL-2 ELISpot assay) showed that CDR-HuCAN20G2was more immunogenic than HE-HuCAN20G2.

TABLE 16 Summary of the mean magnitude (±SD) of positive T cell IL-2secretion responses against the antibodies. Mean Frequency (%) of SampleSI +/−SD Response CDR-HuCAN20G2 2.39 0.52 24 HE-HuCAN20G2 2.63 N/A 5 KLH4.13 1.48 95The data includes borderline responses (SI≧1.90, p<0.05). N/A indicatesno data available.

Interpretation of Results

The proliferation and IL-2 ELISpot assay data show that positive T cellresponses were detected against both test antibodies in a proportion ofthe donors. The overall correlation between proliferation and IL-2ELISpot assays was high (94% for KLH, Table 14) and thus, as in previousstudies, responding donors were defined as those that mounted a positiveresponse to each sample in both IL-2 ELISpot and proliferation assays.Table 14 shows a summary of positive responses against the antibodies inboth proliferation and IL-2 ELISpot assays. Comparison of the dataobtained from the proliferation and IL-2 ELISpot assays showed that theantibodies tested induced homologous frequencies of positive T cellresponses between the assays. All donors produced a positive T cellresponse against PHA in the IL-2 ELISpot assay indicating that cells inthe ex vivo cultures were functional (data not shown). Analysis of thecombined datasets from these two assays revealed that the overallfrequency and magnitude of responses was high for antibody CDR-HuCAN20G2with 24% of donors responding in both proliferation and ELISpot assaysand low for antibody HE-HuCAN20G2 with 5% of donors responding.

Conclusion

The overall correlation between proliferation and IL-2 ELISpot assay washigh, responding donors were defined as those that mounted a positiveresponse to each sample in both assays. Analysis of the combineddatasets from two assays reveals that overall response was high forantibody CDR-huCAN20G2 with 24% of donors responding in both assays andlow for antibody HE-huCAN20G2 with 5% of donors responding. PreviousEpiScreen™ T cell assays with a range of biologics have showed a clearcorrelation between the percentage of donor T cell responses in theassay and the level of immunogenicity observed in clinic, whereas theprotein therapeutics that induced >10% positive response are associatedwith risk of immunogenicity in the clinic. The current study resultsshowed that, in comparison with other protein therapeutics tested inEpiScreen™ assays, antibody CDR-huCAN20G2 would be considered as havinga risk of clinical immunogenicity. In contrast, antibody HE-huCAN20G2would be considered as having a low risk of clinical immunogenicity.

Example 17 In Vivo Efficacy of Humanized CAN20G2 mAbs Against Toxin aChallenge

The in vivo protective efficacy of the two humanized CAN20G2 anti-TcdAmAbs, HE-CAN20G2 and CDR-CAN20G2 were evaluated in the mouse lethaltoxin challenge model (as noted in Example 8 above) by testing a lowdose of antibody. Swiss Webster mice weighing 20-30 g were given 50 ugof mAb or controls at day 0 and allowed to rest. After 24 hrs, the micewere given a lethal dose of TcdA (100 ng). This dose kills 90-100% ofanimals by 24 hours in an unprotected state. The mice were observed fora period of 14 days for clinical symptoms, abnormality and local andsystemic disease. All observations were recorded and the % survival wasdetermined for each treatment group.

Results

As shown in FIGS. 27 and 28, both humanized CAN20G2 mAbs wereefficacious in protecting against toxin A in vivo challenge. HE-CAN20G2conferred better in vivo protection compared to CDA1 and CDR-CAN20G2. Atthe low dose of 0.05 mg/mouse, HE-CAN20G2 recipient mice had a highersurvival rate (90%) compared to those treated with CDA1 (80%) andCDR-CAN20G2 (70%) against TcdA lethal challenge.

Example 18 Pharmacokinetic Analysis of Humanized Antibodies

Pharmacokinetic studies were conducted for CDR-huCAN20G2 andHE-huCAN20G2 in hamster model and rat model. In hamster study, GoldenSyrian hamsters were injected intraperitoneally with 50 mg/kg ofCDR-huCAN20G2. Blood samples were collected at 2 h, 24 h, 48 h, 72 h, 96h, 168 h, 240 h and 336 h post-injection. Control samples were collectedfrom test animal 5 days before injection and sentinel group at differenttime points. The blood samples were centrifuged at 8000 rpm for 10minutes to obtain sera. In rat study, two groups of Sprague-Dawley ratswere instrumented with a femoral vein catheter (FVC) for intravenousdosing and a jugular vein catheter (JVC) for blood collection. Twoantibodies, CDR-huCAN20G2 and HE-huCAN20G2, were injected to each groupof rats at 10 mg/kg dose level via single IV bolus followed by 0.5 mLsaline flush. Blood samples were collected at pre-dose, 0.083, 1, 2, 4,8, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288 and 312 hourspost-dose from the JVC. Whole blood (300 μL) samples were centrifuged at2200×g for 10 minutes to isolate sera.

The antibody concentration in the sera was determined via ELISA. 96-wellELISA plate were coated overnight with goat anti-human IgG, affinitypurified and monkey serum adsorbed (Novus Biologicals) at 1 μg/mL.Plates were washed with PBS-T and blocked with blocking buffer. Theantibody reference standard was diluted in 1% pooled naïve hamster serumto generate a standard curve with a range of 0.098-100 ng/mL. Dilutedtest samples and standards were incubated 1.5 hours at room temperature.Plates were washed and incubated with HRP-goat anti-human IgG, affinitypurified and monkey serum adsorbed (Novus Biologicals), developed withTMB peroxidase substrate system (R&D systems) and stopped with TMBperoxidase stop solution (R&D system). Plates were read on a SpectraMaxplate reader at 450 nm. Antibody concentration in each animal atdifferent time points as calculated using the standard curves.

Results:

For hamster PK study, noncompartmental pharmacokinetic analysis wasperformed using SAS Version 9.2 for Windows, the data are shown in Table17. As indicated, CDR-huCAN20G2 demonstrated a terminal half life around6 days with 50 mg/kg administration dose, which ensured antibodyretention in future efficacy studies.

For rat PK study, noncompartmental pharmacokinetic analysis wasperformed using Watson, version 7.2.0.02 and the data are illustrated inTable 18 and FIGS. 29A and 29B. As indicated, the PK profiles of the twomonoclonal antibodies are very similar. Comparable levels of exposurewere exhibited and metabolism was close to the same rate.

TABLE 17 PK Study of Humanized Antibodies in Hamsters Cmax TmaxAUC_((0-t)) t½ Half-life mAb (μg/mL) (hour) (μg * hour/mL) (hour) CDR-244.9 24 36777.5 166.44 huCAN20G2 50 mg/kg

TABLE 18 PK Study of Humanized Antibodies in Rats t½ AUC_((0-x))Cl_((0-x)) Vd_(ss(0-x)) Half-life mAb μg * hour/mL mL/kg/hr mL/kg (hour)CDR- 14533 0.689 81.8 170 huCAN20G2 10 mg/kg HE- 17500 0.573 70.2 209huCAN20G2 10 mg/kg

While specific aspects of the invention have been described andillustrated, such aspects should be considered illustrative of theinvention only and not as limiting the invention as construed inaccordance with the accompanying claims. All publications and patentapplications cited in this specification are herein incorporated byreference in their entirety for all purposes as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference for all purposes. Although theforegoing invention has been described in some detail by way ofillustration and example for purposes of clarity of understanding, itwill be readily apparent to one of ordinary skill in the art in light ofthe teachings of this invention that certain changes and modificationscan be made thereto without departing from the spirit or scope of theappended claims.

What is claimed is:
 1. An isolated monoclonal antibody, or anantigen-binding portion thereof, comprising a heavy chain region and alight chain region, wherein the heavy chain region comprises threecomplementarity determining regions (CDRs), CDR1, CDR2 and CDR3, havingamino acid sequences about 80% to about 100% homologous to the aminoacid sequences set forth in SEQ ID NOs: 29, 30 and 31, respectively, andwherein the light chain region comprises three CDRs, CDR1, CDR2 andCDR3, having amino acid sequences about 80% to about 100% homologous tothe amino acid sequences set forth in SEQ ID NOs: 21, 22 and 23,respectively.
 2. An isolated monoclonal antibody, or an antigen-bindingportion thereof, that binds to Clostridium difficile (C. difficile)toxin A and comprises a heavy chain region, wherein the heavy chainregion comprises three CDRs, CDR1, CDR2 and CDR3, having amino acidsequences about 80% to about 100% homologous to the amino acid sequencesset forth in SEQ ID NOs: 29, 30 and 31, respectively.
 3. An isolatedmonoclonal antibody, or an antigen-binding portion thereof, that bindsto C. difficile toxin A and comprises a light chain region, wherein thelight chain region comprises three CDRs, CDR1, CDR2 and CDR3, havingamino acid sequences about 80% to about 100% homologous to the aminoacid sequences set forth in SEQ ID NOs: 21, 22 and 23, respectively. 4.The antibody or antigen-binding portion thereof of claim 1, wherein theantibody, or antigen-binding portion thereof, binds to C. difficiletoxin A, and wherein the dissociation constant (K_(D)) of the antibody,or antigen-binding portion thereof, is less than about 1×10⁻¹¹M.
 5. Theantibody or antigen-binding portion thereof of claim 1, wherein theantibody or antigen-binding portion thereof is humanized or chimeric. 6.The antibody or antigen-binding portion thereof of claim 1, wherein theheavy chain region comprises an amino acid sequence about 80% to about100% homologous to the amino acid sequence set forth in SEQ ID NO: 89,and wherein the light chain region comprises an amino acid sequenceabout 80% to about 100% homologous to the amino acid sequence set forthin SEQ ID NO:
 91. 7. The antibody or antigen-binding portion thereof ofclaim 1, wherein the heavy chain region comprises an amino acid sequenceabout 80% to about 100% homologous to the amino acid sequence set forthin SEQ ID NO: 93, and wherein the light chain region comprises an aminoacid sequence about 80% to about 100% homologous to the amino acidsequence set forth in SEQ ID NO:
 95. 8. The antibody or antigen-bindingportion thereof of claim 1, wherein the antibody or antigen-bindingportion thereof is selected from the group consisting of: (a) a wholeimmunoglobulin molecule; (b) an scFv; (c) a Fab fragment; (d) anF(ab′)2; and (e) a disulfide linked Fv.
 9. The antibody orantigen-binding portion thereof of claim 1, wherein the antibody orantigen-binding portion thereof comprises at least one constant domainselected from the group consisting of: a) an IgG constant domain; and(b) an IgA constant domain.
 10. The antibody or antigen-binding portionthereof of claim 1, wherein the antibody or antigen-binding portionthereof binds to fragment 4 of C. difficile toxin A.
 11. An isolatedmonoclonal antibody or an antigen-binding portion thereof, that binds toC. difficile toxin A and comprises a heavy chain variable region,wherein the heavy chain variable region comprises an amino acid sequenceabout 80% to about 100% homologous to the amino acid sequence set forthin SEQ ID NOs: 12, 28, 44 or
 60. 12. An isolated monoclonal antibody, oran antigen-binding portion thereof, that binds to C. difficile toxin Aand comprises a light chain variable region, wherein the light chainvariable region having an amino acid sequence about 80% to about 100%homologous to the amino acid sequence set forth in SEQ ID NOs: 4, 20, 36or
 52. 13. An isolated monoclonal antibody, or an antigen-bindingportion thereof, wherein the antibody, or antigen-binding portionthereof, binds to the same epitope of C. difficile toxin A recognized byan antibody comprising a heavy chain variable region and a light chainvariable region having amino acid sequences about 80% to about 100%homologous to the amino acid sequences set forth in SEQ ID NOs: 28 and20, respectively.
 14. An antibody produced by hybridoma designatedCAN20G2.
 15. A hybridoma designated CAN20G2.
 16. An isolated monoclonalantibody, or an antigen-binding portion thereof, wherein, in an in vivotoxin A challenge experiment, when the antibody, or an antigen-bindingportion thereof, is administered to a mammal at a dosage ranging fromabout 8 mg/kg body weight to about 13 mg/kg body weight about 24 hoursbefore the mammal is exposed to greater than about 100 ng of C.difficile toxin A, the chance of survival for the mammal is greater thanabout 80% within about 7 days.
 17. The antibody or antigen-bindingportion thereof of claim 17, wherein the antibody or antigen-bindingportion thereof comprises a heavy chain region and a light chain region,the heavy chain region comprising three CDRs, CDR1, CDR2, CDR3, havingamino acid sequences about 80% to about 100% homologous to the aminoacid sequences set forth in SEQ ID NOs: 29, 30 and 31, respectively, thelight chain region comprising three CDRs, CDR1, CDR2, CDR3, having aminoacid sequences about 80% to about 100% homologous to the amino acidsequences set forth in SEQ ID NOs: 21, 22 and 23, respectively.
 18. Anisolated monoclonal antibody, or an antigen-binding portion thereof,wherein the antibody, or antigen-binding portion thereof, at aconcentration ranging from about 4 μM to about 17 μM, neutralizesgreater than about 40% of about 150 ng/ml C. difficile toxin A in an invitro neutralization assay.
 19. The antibody or antigen-binding portionthereof of claim 18, wherein the antibody or antigen-binding portionthereof comprises a heavy chain region and a light chain region, theheavy chain region comprising three CDRs, CDR1, CDR2, CDR3, having aminoacid sequences about 80% to about 100% homologous to the amino acidsequences set forth in SEQ ID NOs: 29, 30 and 31, respectively, thelight chain region comprising three CDRs, CDR1, CDR2, CDR3, having aminoacid sequences about 80% to about 100% homologous to the amino acidsequences set forth in SEQ ID NOs: 21, 22 and 23, respectively.
 20. Acomposition comprising the antibody or antigen-binding portion thereofof claim 1, and at least one pharmaceutically acceptable carrier.
 21. Amethod of preventing or treating C. difficile-associated diseasecomprising administering to a subject an effective amount of theantibody or antigen-binding portion thereof of claim
 1. 22. The methodof claim 21, wherein the antibody or antigen-binding portion thereof isadministered intravenously, subcutaneously, intramuscularly ortransdermally.
 23. The method of claim 21, further comprising the stepof administering to the subject a second agent.
 24. The method of claim23, wherein the second agent is a different antibody or fragmentthereof.
 25. The method of claim 23, wherein the second agent is anantibiotic.
 26. The method of claim 25, wherein the antibiotic isvancomycin, metronidazole, or fidaxomicin.